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Sample records for human myosin binding

  1. Three-dimensional stochastic model of actin–myosin binding in the sarcomere lattice

    Energy Technology Data Exchange (ETDEWEB)

    Mijailovich, Srboljub M.; Kayser-Herold, Oliver; Stojanovic, Boban; Nedic, Djordje; Irving, Thomas C.; Geeves, MA (Harvard); (IIT); (U. Kent); (Kragujevac)

    2016-11-18

    The effect of molecule tethering in three-dimensional (3-D) space on bimolecular binding kinetics is rarely addressed and only occasionally incorporated into models of cell motility. The simplest system that can quantitatively determine this effect is the 3-D sarcomere lattice of the striated muscle, where tethered myosin in thick filaments can only bind to a relatively small number of available sites on the actin filament, positioned within a limited range of thermal movement of the myosin head. Here we implement spatially explicit actomyosin interactions into the multiscale Monte Carlo platform MUSICO, specifically defining how geometrical constraints on tethered myosins can modulate state transition rates in the actomyosin cycle. The simulations provide the distribution of myosin bound to sites on actin, ensure conservation of the number of interacting myosins and actin monomers, and most importantly, the departure in behavior of tethered myosin molecules from unconstrained myosin interactions with actin. In addition, MUSICO determines the number of cross-bridges in each actomyosin cycle state, the force and number of attached cross-bridges per myosin filament, the range of cross-bridge forces and accounts for energy consumption. At the macroscopic scale, MUSICO simulations show large differences in predicted force-velocity curves and in the response during early force recovery phase after a step change in length comparing to the two simplest mass action kinetic models. The origin of these differences is rooted in the different fluxes of myosin binding and corresponding instantaneous cross-bridge distributions and quantitatively reflects a major flaw of the mathematical description in all mass action kinetic models. Consequently, this new approach shows that accurate recapitulation of experimental data requires significantly different binding rates, number of actomyosin states, and cross-bridge elasticity than typically used in mass action kinetic models to

  2. Sarcomere lattice geometry influences cooperative myosin binding in muscle.

    Directory of Open Access Journals (Sweden)

    Bertrand C W Tanner

    2007-07-01

    Full Text Available In muscle, force emerges from myosin binding with actin (forming a cross-bridge. This actomyosin binding depends upon myofilament geometry, kinetics of thin-filament Ca(2+ activation, and kinetics of cross-bridge cycling. Binding occurs within a compliant network of protein filaments where there is mechanical coupling between myosins along the thick-filament backbone and between actin monomers along the thin filament. Such mechanical coupling precludes using ordinary differential equation models when examining the effects of lattice geometry, kinetics, or compliance on force production. This study uses two stochastically driven, spatially explicit models to predict levels of cross-bridge binding, force, thin-filament Ca(2+ activation, and ATP utilization. One model incorporates the 2-to-1 ratio of thin to thick filaments of vertebrate striated muscle (multi-filament model, while the other comprises only one thick and one thin filament (two-filament model. Simulations comparing these models show that the multi-filament predictions of force, fractional cross-bridge binding, and cross-bridge turnover are more consistent with published experimental values. Furthermore, the values predicted by the multi-filament model are greater than those values predicted by the two-filament model. These increases are larger than the relative increase of potential inter-filament interactions in the multi-filament model versus the two-filament model. This amplification of coordinated cross-bridge binding and cycling indicates a mechanism of cooperativity that depends on sarcomere lattice geometry, specifically the ratio and arrangement of myofilaments.

  3. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

    Directory of Open Access Journals (Sweden)

    Mirco Müller

    Full Text Available Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

  4. Phosphorylation of human skeletal muscle myosin

    International Nuclear Information System (INIS)

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-01-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30 0 C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with ( 30 P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ

  5. Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

    Science.gov (United States)

    Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J; Polo, Simona

    2016-04-01

    Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.

  6. Phosphorylation of Tropomyosin Extends Cooperative Binding of Myosin Beyond a Single Regulatory Unit

    OpenAIRE

    Rao, Vijay S.; Marongelli, Ellisha N.; Guilford, William H.

    2009-01-01

    Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively ...

  7. A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads.

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    Iwaki, M; Wickham, S F; Ikezaki, K; Yanagida, T; Shih, W M

    2016-12-12

    Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.

  8. Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

    Science.gov (United States)

    Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

    2011-01-01

    Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

  9. A Role for Myosin Va in Human Cytomegalovirus Nuclear Egress.

    Science.gov (United States)

    Wilkie, Adrian R; Sharma, Mayuri; Pesola, Jean M; Ericsson, Maria; Fernandez, Rosio; Coen, Donald M

    2018-03-15

    Herpesviruses replicate and package their genomes into capsids in replication compartments within the nuclear interior. Capsids then move to the inner nuclear membrane for envelopment and release into the cytoplasm in a process called nuclear egress. We previously found that nuclear F-actin is induced upon infection with the betaherpesvirus human cytomegalovirus (HCMV) and is important for nuclear egress and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Despite these and related findings, it has not been shown that any specific motor protein is involved in herpesvirus nuclear egress. In this study, we have investigated whether the host motor protein, myosin Va, could be fulfilling this role. Using immunofluorescence microscopy and coimmunoprecipitation, we observed associations between a nuclear population of myosin Va and the viral major capsid protein, with both concentrating at the periphery of replication compartments. Immunoelectron microscopy showed that nearly 40% of assembled nuclear capsids associate with myosin Va. We also found that myosin Va and major capsid protein colocalize with nuclear F-actin. Importantly, antagonism of myosin Va with RNA interference or a dominant negative mutant revealed that myosin Va is important for the efficient production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Our results lead us to suggest a working model whereby human cytomegalovirus capsids associate with myosin Va for movement from replication compartments to the nuclear periphery during nuclear egress. IMPORTANCE Little is known regarding how newly assembled and packaged herpesvirus capsids move from the nuclear interior to the periphery during nuclear egress. While it has been proposed that an actomyosin-based mechanism facilitates intranuclear movement of alphaherpesvirus capsids, a functional role for

  10. Identification and biochemical analysis of Slac2-c/MyRIP as a Rab27A-, myosin Va/VIIa-, and actin-binding protein.

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    Kuroda, Taruho S; Fukuda, Mitsunori

    2005-01-01

    Slac2-c/MyRIP is a specific Rab27A-binding protein that contains an N-terminal synaptotagmin-like protein (Slp) homology domain (SHD, a newly identified GTP-Rab27A-binding motif), but in contrast to the Slp family proteins, it lacks C-terminal tandem C2 domains. In vitro Slac2-c simultaneously directly interacts with both Rab27A and an actin-based motor protein, myosin Va, via its N-terminal SHD and middle region, respectively, consistent with the fact that the overall structure of Slac2-c is similar to that of Slac2-a/melanophilin, a linker protein between Rab27A and myosin Va in the melanosome transport in melanocytes. Unlike Slac2-a, however, the middle region of Slac2-c interacts with two types of myosins, myosin Va and myosin VIIa. In addition, the most C-terminal part of both Slac2-a and Slac2-c functions as an actin-binding domain: it directly interacts with globular and fibrous actin in vitro, and the actin-binding domain of Slac2-a and Slac2-c colocalizes with actin filaments when it is expressed in living cells (i.e., PC12 cells and mouse melanocytes). In this chapter we describe the methods that have been used to analyze the protein-protein interactions of Slac2-c, specifically with Rab27A, myosin Va/VIIa, and actin.

  11. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

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    Chen, Guokai; Hou, Zhonggang; Gulbranson, Daniel R; Thomson, James A

    2010-08-06

    Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs. Copyright 2010 Elsevier Inc. All rights reserved.

  12. N-terminus of Cardiac Myosin Essential Light Chain Modulates Myosin Step-Size

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    Wang, Yihua; Ajtai, Katalin; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Burghardt, Thomas P.

    2016-01-01

    Muscle myosin cyclically hydrolyzes ATP to translate actin. Ventricular cardiac myosin (βmys) moves actin with three distinct unitary step-sizes resulting from its lever-arm rotation and with step-frequencies that are modulated in a myosin regulation mechanism. The lever-arm associated essential light chain (vELC) binds actin by its 43 residue N-terminal extension. Unitary steps were proposed to involve the vELC N-terminal extension with the 8 nm step engaging the vELC/actin bond facilitating an extra ~19 degrees of lever-arm rotation while the predominant 5 nm step forgoes vELC/actin binding. A minor 3 nm step is the unlikely conversion of the completed 5 to the 8 nm step. This hypothesis was tested using a 17 residue N-terminal truncated vELC in porcine βmys (Δ17βmys) and a 43 residue N-terminal truncated human vELC expressed in transgenic mouse heart (Δ43αmys). Step-size and step-frequency were measured using the Qdot motility assay. Both Δ17βmys and Δ43αmys had significantly increased 5 nm step-frequency and coincident loss in the 8 nm step-frequency compared to native proteins suggesting the vELC/actin interaction drives step-size preference. Step-size and step-frequency probability densities depend on the relative fraction of truncated vELC and relate linearly to pure myosin species concentrations in a mixture containing native vELC homodimer, two truncated vELCs in the modified homodimer, and one native and one truncated vELC in the heterodimer. Step-size and step-frequency, measured for native homodimer and at two or more known relative fractions of truncated vELC, are surmised for each pure species by using a new analytical method. PMID:26671638

  13. Human myosin VIIA responsible for the Usher 1B syndrome: a predicted membrane-associated motor protein expressed in developing sensory epithelia.

    Science.gov (United States)

    Weil, D; Levy, G; Sahly, I; Levi-Acobas, F; Blanchard, S; El-Amraoui, A; Crozet, F; Philippe, H; Abitbol, M; Petit, C

    1996-04-16

    The gene encoding human myosin VIIA is responsible for Usher syndrome type III (USH1B), a disease which associates profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. The reconstituted cDNA sequence presented here predicts a 2215 amino acid protein with a typical unconventional myosin structure. This protein is expected to dimerize into a two-headed molecule. The C terminus of its tail shares homology with the membrane-binding domain of the band 4.1 protein superfamily. The gene consists of 48 coding exons. It encodes several alternatively spliced forms. In situ hybridization analysis in human embryos demonstrates that the myosin VIIA gene is expressed in the pigment epithelium and the photoreceptor cells of the retina, thus indicating that both cell types may be involved in the USH1B retinal degenerative process. In addition, the gene is expressed in the human embryonic cochlear and vestibular neuroepithelia. We suggest that deafness and vestibular dysfunction in USH1B patients result from a defect in the morphogenesis of the inner ear sensory cell stereocilia.

  14. Interaction between cardiac myosin-binding protein C and formin Fhod3.

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    Matsuyama, Sho; Kage, Yohko; Fujimoto, Noriko; Ushijima, Tomoki; Tsuruda, Toshihiro; Kitamura, Kazuo; Shiose, Akira; Asada, Yujiro; Sumimoto, Hideki; Takeya, Ryu

    2018-05-08

    Mutations in cardiac myosin-binding protein C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C has been considered to regulate the cardiac function via cross-bridge arrangement at the C-zone of the myosin-containing A-band, the mechanism by which cMyBP-C functions remains unclear. We identified formin Fhod3, an actin organizer essential for the formation and maintenance of cardiac sarcomeres, as a cMyBP-C-binding protein. The cardiac-specific N-terminal Ig-like domain of cMyBP-C directly interacts with the cardiac-specific N-terminal region of Fhod3. The interaction seems to direct the localization of Fhod3 to the C-zone, since a noncardiac Fhod3 variant lacking the cMyBP-C-binding region failed to localize to the C-zone. Conversely, the cardiac variant of Fhod3 failed to localize to the C-zone in the cMyBP-C-null mice, which display a phenotype of hypertrophic cardiomyopathy. The cardiomyopathic phenotype of cMyBP-C-null mice was further exacerbated by Fhod3 overexpression with a defect of sarcomere integrity, whereas that was partially ameliorated by a reduction in the Fhod3 protein levels, suggesting that Fhod3 has a deleterious effect on cardiac function under cMyBP-C-null conditions where Fhod3 is aberrantly mislocalized. Together, these findings suggest the possibility that Fhod3 contributes to the pathogenesis of cMyBP-C-related cardiomyopathy and that Fhod3 is critically involved in cMyBP-C-mediated regulation of cardiac function via direct interaction.

  15. Myosin content of individual human muscle fibers isolated by laser capture microdissection.

    Science.gov (United States)

    Stuart, Charles A; Stone, William L; Howell, Mary E A; Brannon, Marianne F; Hall, H Kenton; Gibson, Andrew L; Stone, Michael H

    2016-03-01

    Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. Copyright © 2016 the American Physiological Society.

  16. Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.

    Science.gov (United States)

    Sato, Osamu; Komatsu, Satoshi; Sakai, Tsuyoshi; Tsukasaki, Yoshikazu; Tanaka, Ryosuke; Mizutani, Takeomi; Watanabe, Tomonobu M; Ikebe, Reiko; Ikebe, Mitsuo

    2017-06-30

    Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s -1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s -1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Random myosin loss along thick-filaments increases myosin attachment time and the proportion of bound myosin heads to mitigate force decline in skeletal muscle

    Science.gov (United States)

    Tanner, Bertrand C.W.; McNabb, Mark; Palmer, Bradley M.; Toth, Michael J.; Miller, Mark S.

    2014-01-01

    Diminished skeletal muscle performance with aging, disuse, and disease may be partially attributed to the loss of myofilament proteins. Several laboratories have found a disproportionate loss of myosin protein content relative to other myofilament proteins, but due to methodological limitations, the structural manifestation of this protein loss is unknown. To investigate how variations in myosin content affect ensemble cross-bridge behavior and force production we simulated muscle contraction in the half-sarcomere as myosin was removed either i) uniformly, from the Z-line end of thick-filaments, or ii) randomly, along the length of thick-filaments. Uniform myosin removal decreased force production, showing a slightly steeper force-to-myosin content relationship than the 1:1 relationship that would be expected from the loss of cross-bridges. Random myosin removal also decreased force production, but this decrease was less than observed with uniform myosin loss, largely due to increased myosin attachment time (ton) and fractional cross-bridge binding with random myosin loss. These findings support our prior observations that prolonged ton may augment force production in single fibers with randomly reduced myosin content from chronic heart failure patients. These simulation also illustrate that the pattern of myosin loss along thick-filaments influences ensemble cross-bridge behavior and maintenance of force throughout the sarcomere. PMID:24486373

  18. An endogenously produced fragment of cardiac myosin-binding protein C is pathogenic and can lead to heart failure.

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    Razzaque, Md Abdur; Gupta, Manish; Osinska, Hanna; Gulick, James; Blaxall, Burns C; Robbins, Jeffrey

    2013-08-16

    A stable 40-kDa fragment is produced from cardiac myosin-binding protein C when the heart is stressed using a stimulus, such as ischemia-reperfusion injury. Elevated levels of the fragment can be detected in the diseased mouse and human heart, but its ability to interfere with normal cardiac function in the intact animal is unexplored. To understand the potential pathogenicity of the 40-kDa fragment in vivo and to investigate the molecular pathways that could be targeted for potential therapeutic intervention. We generated cardiac myocyte-specific transgenic mice using a Tet-Off inducible system to permit controlled expression of the 40-kDa fragment in cardiomyocytes. When expression of the 40-kDa protein is induced by crossing the responder animals with tetracycline transactivator mice under conditions in which substantial quantities approximating those observed in diseased hearts are reached, the double-transgenic mice subsequently experience development of sarcomere dysgenesis and altered cardiac geometry, and the heart fails between 12 and 17 weeks of age. The induced double-transgenic mice had development of cardiac hypertrophy with myofibrillar disarray and fibrosis, in addition to activation of pathogenic MEK-ERK pathways. Inhibition of MEK-ERK signaling was achieved by injection of the mitogen-activated protein kinase (MAPK)/ERK inhibitor U0126. The drug effectively improved cardiac function, normalized heart size, and increased probability of survival. These results suggest that the 40-kDa cardiac myosin-binding protein C fragment, which is produced at elevated levels during human cardiac disease, is a pathogenic fragment that is sufficient to cause hypertrophic cardiomyopathy and heart failure.

  19. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients

    KAUST Repository

    Conti, Antonio

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS\\'s pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS. © 2013 Elsevier B.V.

  20. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    Science.gov (United States)

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-09

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

  1. Azidoblebbistatin, a photoreactive myosin inhibitor

    Science.gov (United States)

    Képiró, Miklós; Várkuti, Boglárka H.; Bodor, Andrea; Hegyi, György; Drahos, László; Kovács, Mihály; Málnási-Csizmadia, András

    2012-01-01

    Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC50 ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC50 = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions. PMID:22647605

  2. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients.

    Science.gov (United States)

    Conti, Antonio; Riva, Nilo; Pesca, Mariasabina; Iannaccone, Sandro; Cannistraci, Carlo V; Corbo, Massimo; Previtali, Stefano C; Quattrini, Angelo; Alessio, Massimo

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS's pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS. © 2013 Elsevier B.V. All rights reserved.

  3. Metastasis-associated protein Mts1 (S100A4) inhibits CK2-mediated phosphorylation and self-assembly of the heavy chain of nonmuscle myosin

    DEFF Research Database (Denmark)

    Kriajevska, M; Bronstein, I B; Scott, D J

    2000-01-01

    a regulatory role in the myosin assembly. In the presence of calcium, Mts1 binds at the C-terminal end of the myosin heavy chain close to the site of phosphorylation by protein kinase CK2 (Ser1944). In the present study, we have shown that interaction of Mts1 with the human platelet myosin or C...

  4. Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

    International Nuclear Information System (INIS)

    Minoda, Hiroki; Okabe, Tatsuhiro; Inayoshi, Yuhri; Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru; Katayama, Eisaku; Wakabayashi, Takeyuki; Akimoto, Tsuyoshi; Sugi, Haruo

    2011-01-01

    Research highlights: → We succeeded in recording structural changes of hydrated myosin cross-bridges. → We succeeded in position-marking the cross-bridges with site-directed antibodies. → We recorded cross-bridge movement at different regions in individual cross-bridge. → The movement was smallest at the cross-bridge-subfragment two boundary. → The results provide evidence for the cross-bridge lever arm mechanism. -- Abstract: Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

  5. Myosin-1A Targets to Microvilli Using Multiple Membrane Binding Motifs in the Tail Homology 1 (TH1) Domain*

    Science.gov (United States)

    Mazerik, Jessica N.; Tyska, Matthew J.

    2012-01-01

    One of the most abundant components of the enterocyte brush border is the actin-based monomeric motor, myosin-1a (Myo1a). Within brush border microvilli, Myo1a carries out a number of critical functions at the interface between membrane and actin cytoskeleton. Proper physiological function of Myo1a depends on its ability to bind to microvillar membrane, an interaction mediated by a C-terminal tail homology 1 (TH1) domain. However, little is known about the mechanistic details of the Myo1a-TH1/membrane interaction. Structure-function analysis of Myo1a-TH1 targeting in epithelial cells revealed that an N-terminal motif conserved among class I myosins and a C-terminal motif unique to Myo1a-TH1 are both required for steady state microvillar enrichment. Purified Myo1a bound to liposomes composed of phosphatidylserine and phosphoinositol 4,5-bisphosphate, with moderate affinity in a charge-dependent manner. Additionally, peptides of the N- and C-terminal regions required for targeting were able to compete with Myo1a for binding to highly charged liposomes in vitro. Single molecule total internal reflection fluorescence microscopy showed that these motifs are also necessary for slowing the membrane detachment rate in cells. Finally, Myo1a-TH1 co-localized with both lactadherin-C2 (a phosphatidylserine-binding protein) and PLCδ1-PH (a phosphoinositol 4,5-bisphosphate-binding protein) in microvilli, but only lactaderin-C2 expression reduced brush border targeting of Myo1a-TH1. Together, our results suggest that Myo1a targeting to microvilli is driven by membrane binding potential that is distributed throughout TH1 rather than localized to a single motif. These data highlight the diversity of mechanisms that enable different class I myosins to target membranes in distinct biological contexts. PMID:22367206

  6. Multidimensional structure-function relationships in human β-cardiac myosin from population-scale genetic variation

    NARCIS (Netherlands)

    Homburger, J.R. (Julian R.); Green, E.M. (Eric M.); Caleshu, C. (Colleen); Sunitha, M.S. (Margaret S.); Taylor, R.E. (Rebecca E.); Ruppel, K.M. (Kathleen M.); Metpally, R.P.R. (Raghu Prasad Rao); S.D. Colan (Steven); M. Michels (Michelle); Day, S.M. (Sharlene M.); I. Olivotto (Iacopo); Bustamante, C.D. (Carlos D.); Dewey, F.E. (Frederick E.); Ho, C.Y. (Carolyn Y.); Spudich, J.A. (James A.); Ashley, E.A. (Euan A.)

    2016-01-01

    textabstractMyosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac

  7. Evolutionary traces decode molecular mechanism behind fast pace of myosin XI

    Directory of Open Access Journals (Sweden)

    Syamaladevi Divya P

    2011-09-01

    Full Text Available Abstract Background Cytoplasmic class XI myosins are the fastest processive motors known. This class functions in high-velocity cytoplasmic streaming in various plant cells from algae to angiosperms. The velocities at which they process are ten times faster than its closest class V homologues. Results To provide sequence determinants and structural rationale for the molecular mechanism of this fast pace myosin, we have compared the sequences from myosin class V and XI through Evolutionary Trace (ET analysis. The current study identifies class-specific residues of myosin XI spread over the actin binding site, ATP binding site and light chain binding neck region. Sequences for ET analysis were accumulated from six plant genomes, using literature based text search and sequence searches, followed by triple validation viz. CDD search, string-based searches and phylogenetic clustering. We have identified nine myosin XI genes in sorghum and seven in grape by sequence searches. Both the plants possess one gene product each belonging to myosin type VIII as well. During this process, we have re-defined the gene boundaries for three sorghum myosin XI genes using fgenesh program. Conclusion Molecular modelling and subsequent analysis of putative interactions involving these class-specific residues suggest a structural basis for the molecular mechanism behind high velocity of plant myosin XI. We propose a model of a more flexible switch I region that contributes to faster ADP release leading to high velocity movement of the algal myosin XI.

  8. Distribution and evolution of stable single α-helices (SAH domains in myosin motor proteins.

    Directory of Open Access Journals (Sweden)

    Dominic Simm

    Full Text Available Stable single-alpha helices (SAHs are versatile structural elements in many prokaryotic and eukaryotic proteins acting as semi-flexible linkers and constant force springs. This way SAH-domains function as part of the lever of many different myosins. Canonical myosin levers consist of one or several IQ-motifs to which light chains such as calmodulin bind. SAH-domains provide flexibility in length and stiffness to the myosin levers, and may be particularly suited for myosins working in crowded cellular environments. Although the function of the SAH-domains in human class-6 and class-10 myosins has well been characterised, the distribution of the SAH-domain in all myosin subfamilies and across the eukaryotic tree of life remained elusive. Here, we analysed the largest available myosin sequence dataset consisting of 7919 manually annotated myosin sequences from 938 species representing all major eukaryotic branches using the SAH-prediction algorithm of Waggawagga, a recently developed tool for the identification of SAH-domains. With this approach we identified SAH-domains in more than one third of the supposed 79 myosin subfamilies. Depending on the myosin class, the presence of SAH-domains can range from a few to almost all class members indicating complex patterns of independent and taxon-specific SAH-domain gain and loss.

  9. Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

    OpenAIRE

    Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

    2011-01-01

    Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic bindin...

  10. The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization.

    Science.gov (United States)

    He, Qiuxia; Liu, Kechun; Tian, Zhenjun; Du, Shao Jun

    2015-01-01

    Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation.

  11. Preparation of human cardiac anti-myosin: a review

    International Nuclear Information System (INIS)

    Okada, H.; Souza, I.T.T.

    1990-01-01

    The present communication is a review of the physicochemical characterization and immunological properties of myosin isolated from the cardiac muscle, the production of monoclonal antibody anti-myosin, the radiolabeling of this antibody and its applications as radiopharmaceuticals to imaging myocardial infarcts. The classical example of radioimmunologic diagnosis of non malignant tissues is the detection of myocardial infarction by radiolabeled antibodies to myosin. (author)

  12. Calcium and cargoes as regulators of myosin 5a activity

    International Nuclear Information System (INIS)

    Sellers, James R.; Thirumurugan, Kavitha; Sakamoto, Takeshi; Hammer, John A.; Knight, Peter J.

    2008-01-01

    Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins

  13. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Gomibuchi, Yuki [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Uyeda, Taro Q.P. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Department of Judo Therapy, Faculty of Medical Technology, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan)

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA

  14. Molecular cloning and complete nucleotide sequence of a human ventricular myosin light chain 1

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, E; Shi, Q W; Floroff, M; Mickle, D A.G.; Wu, T W; Olley, P M; Jackowski, G

    1988-03-25

    Human ventricular plasmid library was constructed. The library was screened with the oligonucleotide probe (17-mer) corresponding to a conserve region of myosin light chain 1 near the carboxy terminal. Full length cDNA recombinant plasmid containing 1100 bp insert was isolated. RNA blot hybridization with this insert detected a message of approximately 1500 bp corresponding to the size of VLCl and mRNA. Complete nucleotide sequence of the coding region was determined in M13 subclones using dideoxy chain termination method. With the isolation of this clone (pCD HLVCl), the publication of the complete nucleotide sequence of HVLCl and the predicted secondary structure of this protein will aid in understanding of the biochemistry of myosin and its function in contraction, the evolution of myosin light genes and the genetic, developmental and physiological regulation of myosin genes.

  15. Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

    Science.gov (United States)

    Lee, Eunhee; Stafford, Walter F

    2015-01-01

    Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

  16. Regulation of actomyosin ATPase activity by troponin-tropomyosin: effect of the binding of the myosin subfragment 1 (S-1) ATP complex

    International Nuclear Information System (INIS)

    Greene, L.E.; Williams, D.L. Jr.; Eisenberg, E.

    1987-01-01

    In the authors' model of regulation, the observed lack of cooperativity in the binding of myosin subfragment 1 (S-1) with bound ATP to the troponin-tropomyosin-actin complex (regulated actin) is explained by S-1 ATP having about the same affinity for the conformation of the regulated actin that activates the myosin ATPase activity (turned-on form) and the conformation that does not activate the myosin ATPase activity (turned-off form). This predicts that, in the absence of Ca 2+ , S-1 ATP should not turn on the regulated actin filament. In the present study, they tested this prediction by using either unmodified S-1 or S-1 chemically modified with N,N'-p-phenylenedimaleimide (pPDM S-1) so that functionally it acts like S-1 ATP, although it does not hydrolyze ATP. [ 14 C]pPDM and [ 32 P]ATP were used as tracers. They found that, in the absence of Ca 2+ , neither S-1 ATP nor pPDM S-1 ATP significantly turns on the ATPase activity of the regulated complex of actin and S-1 (acto S-1). In contrast, in the presence of Ca 2+ , pPDM S-1 ATP binding almost completely turns on the regulated acto S-1 ATPase activity. These results can be explained by their original cooperativity model, with pPDM S-1 ATP binding only ≅ 2 fold more strongly to the turned-on form that to the turned-off form of regulated actin. However, the results are not consistent with our alternative model, which predicts that if pPDM S-1 ATP binds to actin in the absence of Ca 2+ but does not turn on the ATPase activity, then it should also turn on the ATPase activity in the presence of Ca 2+

  17. Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

    International Nuclear Information System (INIS)

    Vu, N.D.; Wagner, P.D.

    1987-01-01

    Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of 32 P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca 2+ - and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1. Although this subfragment 1 contained intact light chains, its actin-activated ATPase activity was not affected by light chain phosphorylation

  18. Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner.

    Directory of Open Access Journals (Sweden)

    Li Wang

    Full Text Available Based on our recent finding that cardiac myosin binding protein C (cMyBP-C phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302, DAD (Asp273-Ala282-Asp302, SAS (Ser273-Ala282-Ser302, and t/t (cMyBP-C null genotypes, and the results were compared to transgenic mice expressing wide-type (WT cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi, and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc, and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.

  19. Cardiac Myosin Binding Protein-C Autoantibodies Are Potential Early Indicators of Cardiac Dysfunction and Patient Outcome in Acute Coronary Syndrome

    Directory of Open Access Journals (Sweden)

    Thomas L. Lynch, IVPhD

    2017-04-01

    Full Text Available Summary: The degradation and release of cardiac myosin binding protein-C (cMyBP-C upon cardiac damage may stimulate an inflammatory response and autoantibody (AAb production. We determined whether the presence of cMyBP-C-AAbs associated with adverse cardiac function in cardiovascular disease patients. Importantly, cMyBP-C-AAbs were significantly detected in acute coronary syndrome patient sera upon arrival to the emergency department, particularly in ST-segment elevation myocardial infarction patients. Patients positive for cMyBP-C-AAbs had reduced left ventricular ejection fraction and elevated levels of clinical biomarkers of myocardial infarction. We conclude that cMyBP-C-AAbs may serve as early predictive indicators of deteriorating cardiac function and patient outcome in acute coronary syndrome patients prior to the infarction. Key Words: acute myocardial infarction, autoantibodies, cardiac myosin binding protein-c, cardiomyopathy

  20. Association of cardiac myosin binding protein-C with the ryanodine receptor channel: putative retrograde regulation?

    Science.gov (United States)

    Stanczyk, Paulina J; Seidel, Monika; White, Judith; Viero, Cedric; George, Christopher H; Zissimopoulos, Spyros; Lai, F Anthony

    2018-06-21

    The cardiac muscle ryanodine receptor-Ca 2+ release channel (RyR2) constitutes the sarcoplasmic reticulum (SR) Ca 2+ efflux mechanism that initiates myocyte contraction, while cardiac myosin binding protein-C (cMyBP-C) mediates regulation of acto-myosin cross-bridge cycling. In this report, we provide the first evidence for the presence of direct interaction between these two proteins, forming a RyR2:cMyBP-C complex. The C-terminus of cMyBP-C binds with the RyR2 N-terminus in mammalian cells and is not mediated by a fibronectin-like domain. Notably, we detected complex formation between both recombinant cMyBP-C and RyR2, as well as with the native proteins in cardiac tissue. Cellular Ca 2+ dynamics in HEK293 cells is altered upon co-expression of cMyBP-C and RyR2, with lowered frequency of RyR2-mediated spontaneous Ca 2+ oscillations, suggesting cMyBP-C exerts a potential inhibitory effect on RyR2-dependent Ca 2+ release. Discovery of a functional RyR2 association with cMyBP-C provides direct evidence for a putative mechanistic link between cytosolic soluble cMyBP-C and SR-mediated Ca 2+ release, via RyR2. Importantly, this interaction may have clinical relevance to the observed cMyBP-C and RyR2 dysfunction in cardiac pathologies, such as hypertrophic cardiomyopathy. © 2018. Published by The Company of Biologists Ltd.

  1. Myosin isoform determines the conformational dynamics and cooperativity of actin filaments in the strongly bound actomyosin complex

    Science.gov (United States)

    Prochniewicz, Ewa; Chin, Harvey F.; Henn, Arnon; Hannemann, Diane E.; Olivares, Adrian O.; Thomas, David D.; De La Cruz, Enrique M.

    2010-01-01

    SUMMARY We have used transient phosphorescence anisotropy (TPA) to detect the microsecond rotational dynamics of erythrosin iodoacetamide (ErIA)-labeled actin strongly bound to single-headed fragments of muscle myosin (muscle S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at sub-stoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects; muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly, and muscle myosin more slowly, to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound ErIA; while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex is determined by the isoform of the bound myosin, in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells. PMID:19962990

  2. Preparation of monoclonal antibodies against cardiac myosin and some radiolabelling studies

    International Nuclear Information System (INIS)

    Bapat, K.; Venkatesh, M.; Pillai, M.R.A.; Sarma, H.D.; Sainis, K.B.

    1998-01-01

    Monoclonal antibodies were raised against myosin, a specific indicator of myocardial infarction and labelled with 125 I and 99m Tc. Human cardiac myosin was isolated from normal human heart and was used for raising the monoclonal antibodies by the hybridoma technique. Antibody producing clones were identified by ELISA and cloning was done by the limiting dilution technique. Of the 13 clones obtained, 4 were deemed suitable for further studies. The antibodies were grown in ascites, purified, isotyped and their cross reactions with other forms of myosin were estimated. All the clones showed negligible cross reaction with rabbit myosin, but reacted to different extents with bovine skeletal myosin. The most avid antibody Mab-4G4 was chosen for further labelling studies. Mab-4G4 was labelled with 125 I using different oxidising agents such as iodogen, chloramine-T and lactoperoxidase. Purified radioiodinated antibody with radiochemical purity >95% could be obtained by gel filtration. Immunoreactivity was retained as tested by binding to myosin immobilised on a solid support. Mab-4G4 was also labelled with 99m Tc using stannous tartrate as the reducing agent. Radiolabelling yield was ∼60%, the purity was >95% and the immunoreactivity was retained. Both the labelled preparations were tested for bio-distribution in normal and infarcted rats. The activity accumulation in the infarcted region was ∼ 1.5 and 3.5 times as that in normal heart muscle for 125 I and 99m Tc labelled Mab-4G4 respectively. The major problem with the iodinated antibody was the in vivo deiodination resulting in very high percentage of activity in the thyroid. Although the fraction of the total activity associated with the infarcted heart is not very impressive, the fact that the activities with the infarcted and normal hearths are significantly different is heartening. With further optimisation of labelling and use of F(ab)'2 fragments, better delineation of the infarct sites is aspired. (author)

  3. Structural insight into the UNC-45–myosin complex

    DEFF Research Database (Denmark)

    Fratev, Filip; Jonsdottir, Svava Osk; Pajeva, Ilza

    2013-01-01

    The UNC-45 chaperone protein interacts with and affects the folding, stability, and the ATPase activity of myosins. It plays a critical role in the cardiomyopathy development and in the breast cancer tumor growth. Here we propose the first structural model of the UNC-45–myosin complex using various...... is mainly stabilized by electrostatic interactions. Remarkably, the contact surface area is similar to that of the myosinactin complex. A significant interspecies difference in the myosin binding epitope is observed. Our results reveal the structural basis of MYH7 exons 15–16 hypertrophic cardiomyopathy...... mutations and provide directions for drug targeting. © 2013 Wiley Periodicals, Inc....

  4. Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice.

    Science.gov (United States)

    Orfanos, Zacharias; Sparrow, John C

    2013-01-01

    During muscle development myosin molecules form symmetrical thick filaments, which integrate with the thin filaments to produce the regular sarcomeric lattice. In Drosophila indirect flight muscles (IFMs) the details of this process can be studied using genetic approaches. The weeP26 transgenic line has a GFP-encoding exon inserted into the single Drosophila muscle myosin heavy chain gene, Mhc. The weeP26 IFM sarcomeres have a unique MHC-GFP-labelling pattern restricted to the sarcomere core, explained by non-translation of the GFP exon following alternative splicing. Characterisation of wild-type IFM MHC mRNA confirmed the presence of an alternately spliced isoform, expressed earlier than the major IFM-specific isoform. The two wild-type IFM-specific MHC isoforms differ by the presence of a C-terminal 'tailpiece' in the minor isoform. The sequential expression and assembly of these two MHCs into developing thick filaments suggest a role for the tailpiece in initiating A-band formation. The restriction of the MHC-GFP sarcomeric pattern in weeP26 is lifted when the IFM lack the IFM-specific myosin binding protein flightin, suggesting that it limits myosin dissociation from thick filaments. Studies of flightin binding to developing thick filaments reveal a progressive binding at the growing thick filament tips and in a retrograde direction to earlier assembled, proximal filament regions. We propose that this flightin binding restricts myosin molecule incorporation/dissociation during thick filament assembly and explains the location of the early MHC isoform pattern in the IFM A-band.

  5. Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells.

    Science.gov (United States)

    Kampourakis, Thomas; Yan, Ziqian; Gautel, Mathias; Sun, Yin-Biao; Irving, Malcolm

    2014-12-30

    Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.

  6. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    International Nuclear Information System (INIS)

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    2011-01-01

    Highlights: ► Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. ► Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. ► It is a widely believed that MYL2 is a poor substrate for smMLCK. ► In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. ► Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca 2+ sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis–Menten V max and K M for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.

  7. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    Energy Technology Data Exchange (ETDEWEB)

    Josephson, Matthew P.; Sikkink, Laura A. [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Penheiter, Alan R. [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States); Burghardt, Thomas P., E-mail: burghardt@mayo.edu [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905 (United States); Ajtai, Katalin [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  8. Remodeling of repolarization and arrhythmia susceptibility in a myosin-binding protein C knockout mouse model.

    Science.gov (United States)

    Toib, Amir; Zhang, Chen; Borghetti, Giulia; Zhang, Xiaoxiao; Wallner, Markus; Yang, Yijun; Troupes, Constantine D; Kubo, Hajime; Sharp, Thomas E; Feldsott, Eric; Berretta, Remus M; Zalavadia, Neil; Trappanese, Danielle M; Harper, Shavonn; Gross, Polina; Chen, Xiongwen; Mohsin, Sadia; Houser, Steven R

    2017-09-01

    Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases and among the leading causes of sudden cardiac death (SCD) in the young. The cellular mechanisms leading to SCD in HCM are not well known. Prolongation of the action potential (AP) duration (APD) is a common feature predisposing hypertrophied hearts to SCD. Previous studies have explored the roles of inward Na + and Ca 2+ in the development of HCM, but the role of repolarizing K + currents has not been defined. The objective of this study was to characterize the arrhythmogenic phenotype and cellular electrophysiological properties of mice with HCM, induced by myosin-binding protein C (MyBPC) knockout (KO), and to test the hypothesis that remodeling of repolarizing K + currents causes APD prolongation in MyBPC KO myocytes. We demonstrated that MyBPC KO mice developed severe hypertrophy and cardiac dysfunction compared with wild-type (WT) control mice. Telemetric electrocardiographic recordings of awake mice revealed prolongation of the corrected QT interval in the KO compared with WT control mice, with overt ventricular arrhythmias. Whole cell current- and voltage-clamp experiments comparing KO with WT mice demonstrated ventricular myocyte hypertrophy, AP prolongation, and decreased repolarizing K + currents. Quantitative RT-PCR analysis revealed decreased mRNA levels of several key K + channel subunits. In conclusion, decrease in repolarizing K + currents in MyBPC KO ventricular myocytes contributes to AP and corrected QT interval prolongation and could account for the arrhythmia susceptibility. NEW & NOTEWORTHY Ventricular myocytes isolated from the myosin-binding protein C knockout hypertrophic cardiomyopathy mouse model demonstrate decreased repolarizing K + currents and action potential and QT interval prolongation, linking cellular repolarization abnormalities with arrhythmia susceptibility and the risk for sudden cardiac death in hypertrophic cardiomyopathy. Copyright © 2017

  9. Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

    International Nuclear Information System (INIS)

    Wu, Shenping; Liu, Jun; Reedy, Mary C.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A.

    2010-01-01

    Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the 'target zone', situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77 o /12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127 o range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from

  10. Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

    Directory of Open Access Journals (Sweden)

    Shenping Wu

    2010-09-01

    Full Text Available Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very

  11. Excessive Myosin Activity in Mbs Mutants Causes Photoreceptor Movement Out of the Drosophila Eye Disc Epithelium

    OpenAIRE

    Lee, Arnold; Treisman, Jessica E.

    2004-01-01

    Neuronal cells must extend a motile growth cone while maintaining the cell body in its original position. In migrating cells, myosin contraction provides the driving force that pulls the rear of the cell toward the leading edge. We have characterized the function of myosin light chain phosphatase, which down-regulates myosin activity, in Drosophila photoreceptor neurons. Mutations in the gene encoding the myosin binding subunit of this enzyme cause photoreceptors to drop out of the eye disc e...

  12. Topology of interaction between titin and myosin thick filaments.

    Science.gov (United States)

    Kellermayer, Miklós; Sziklai, Dominik; Papp, Zsombor; Decker, Brennan; Lakatos, Eszter; Mártonfalvi, Zsolt

    2018-05-05

    Titin is a giant protein spanning between the Z- and M-lines of the sarcomere. In the A-band titin is associated with the myosin thick filament. It has been speculated that titin may serve as a blueprint for thick-filament formation due to the super-repeat structure of its A-band domains. Accordingly, titin might provide a template that determines the length and structural periodicity of the thick filament. Here we tested the titin ruler hypothesis by mixing titin and myosin at in situ stoichiometric ratios (300 myosins per 12 titins) in buffers of different ionic strength (KCl concentration range 100-300 mM). The topology of the filamentous complexes was investigated with atomic force microscopy. We found that the samples contained distinct, segregated populations of titin molecules and myosin thick filaments. We were unable to identify complexes in which myosin molecules were regularly associated to either mono- or oligomeric titin in either relaxed or stretched states of the titin filaments. Thus, the electrostatically driven self-association is stronger in both myosin and titin than their binding to each other, and it is unlikely that titin functions as a geometrical template for thick-filament formation. However, when allowed to equilibrate configurationally, long myosin thick filaments appeared with titin oligomers attached to their surface. The titin meshwork formed on the thick-filament surface may play a role in controlling thick-filament length by regulating the structural dynamics of myosin molecules and placing a mechanical limit on the filament length. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Direct observation of the myosin Va recovery stroke that contributes to unidirectional stepping along actin.

    Directory of Open Access Journals (Sweden)

    Katsuyuki Shiroguchi

    2011-04-01

    Full Text Available Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed "strokes"; the "power stroke" is the force-generating swinging of the myosin light chain-binding "neck" domain relative to the motor domain "head" while bound to actin; the "recovery stroke" is the necessary initial motion that primes, or "cocks," myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a "hand over hand" mechanism in which the trailing head detaches and steps forward ∼72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ∼40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥ 5 k(BT of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery

  14. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Directory of Open Access Journals (Sweden)

    Ivanna Ihnatovych

    Full Text Available Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C. Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate- cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  15. Actin and myosin contribute to mammalian mitochondrial DNA maintenance

    Science.gov (United States)

    Reyes, A.; He, J.; Mao, C. C.; Bailey, L. J.; Di Re, M.; Sembongi, H.; Kazak, L.; Dzionek, K.; Holmes, J. B.; Cluett, T. J.; Harbour, M. E.; Fearnley, I. M.; Crouch, R. J.; Conti, M. A.; Adelstein, R. S.; Walker, J. E.; Holt, I. J.

    2011-01-01

    Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and β-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of β-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some β-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance. PMID:21398640

  16. Technetium-99m labeling of antibodies to cardiac myosin Fab and to human fibrinogen

    International Nuclear Information System (INIS)

    Khaw, B.A.; Strauss, H.W.; Carvalho, A.; Locke, E.; Gold, H.K.; Haber, E.

    1982-01-01

    A method of labeling biologically active labile macromolecules, such as human fibrinogen (HF) and anticardiac-myosin Fab (AM-Fab), with Tc-99m at neutral pH was developed. This method uses dithionite reduction of pertechnetate and subsequent labeling to test the method with acid-labile macromolecules. Complexes of diethylene triamine pentaacetic acid with macromolecules such as human fibrinogen (D-HF) and anticardiac-myosin Fab (D-AM-Fab) were labeled and utilized in in vitro and in vivo studies. In biodistribution studies, the Tc-99m D-HF had a two-component blood clearance (half-times 1 hr and 15 hr) and was 80-88% coagulable. The Tc-99m AM-Fab retained its immunoreactivity as tested by affinity chromatography; also during in vivo localization in experimental myocardial infarction. This labeling technique provides an easy and efficient approach to the Tc-99m labeling of other biologically active and acid-labile macromolecules

  17. Human Usher 1B/mouse shaker-1: the retinal phenotype discrepancy explained by the presence/absence of myosin VIIA in the photoreceptor cells.

    Science.gov (United States)

    el-Amraoui, A; Sahly, I; Picaud, S; Sahel, J; Abitbol, M; Petit, C

    1996-08-01

    Usher syndrome type 1 (USH1) associates severe congenital deafness, vestibular dysfunction and progressive retinitis pigmentosa leading to blindness. The gene encoding myosin VIIA is responsible for USH1B. Mutations in the murine orthologous gene lead to the shaker-1 phenotype, which manifests cochlear and vestibular dysfunction, without any retinal defect. To address this phenotypic discrepancy, the expression of myosin VIIA in retinal cells was analyzed in human and mouse during embryonic development and adult life. In the human embryo, myosin VIIA was present first in the pigment epithelium cells, and later in these cells as well as in the photoreceptor cells. In the adult human retina, myosin VIIA was present in both cell types. In contrast, in mouse, only pigment epithelium cells expressed the protein throughout development and adult life. Myosin VIIA was also found to be absent in the photoreceptor cells of other rodents (rat and guinea-pig), whereas these cells expressed the protein in amphibians, avians and primates. These observations suggest that retinitis pigmentosa of USH1B results from a primary rod and cone defect. The USH1B/shaker-1 paradigm illustrates a species-specific cell pattern of gene expression as a possible cause for the discrepancy between phenotypes involving defective orthologous genes in man and mouse. Interestingly, in the photoreceptor cells, myosin VIIA is mainly localized in the inner and base of outer segments as well as in the synaptic ending region where it is co-localized with the synaptic vesicles. Therefore, we suggest that myosin VIIA might play a role in the trafficking of ribbon-synaptic vesicle complexes and the renewal processes of the outer photoreceptor disks.

  18. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    Science.gov (United States)

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

  19. Functions of myosin motors tailored for parasitism

    DEFF Research Database (Denmark)

    Mueller, Christina; Graindorge, Arnault; Soldati-Favre, Dominique

    2017-01-01

    Myosin motors are one of the largest protein families in eukaryotes that exhibit divergent cellular functions. Their roles in protozoans, a diverse group of anciently diverged, single celled organisms with many prominent members known to be parasitic and to cause diseases in human and livestock......, are largely unknown. In the recent years many different approaches, among them whole genome sequencing, phylogenetic analyses and functional studies have increased our understanding on the distribution, protein architecture and function of unconventional myosin motors in protozoan parasites. In Apicomplexa......, myosins turn out to be highly specialized and to exhibit unique functions tailored to accommodate the lifestyle of these parasites....

  20. /sup 99m/Tc labeling of antibodies to cardiac myosin Fab and to human fibrinogen

    International Nuclear Information System (INIS)

    Khaw, B.A.; Strauss, H.W.; Carvalho, A.; Locke, E.; Gold, H.K.; Haber, E.

    1982-01-01

    We have developed a method of labeling biologically active labile macromolecules, such as human fibrinogen (HF) and anticardiac-myosin Fab (AM-Fab), with /sup 99m/Tc at neutral pH. This method uses dithionite reduction of pertechnetate and subsequent labeling, to test the method with acid-labile macromolecules. Complexes of diethylene triamine pentaacetic acid with macromolecules such as human fibrinogen (D-HF) and anticardiac-myosin Fab (D-AM-Fab) were labeled and utilized in in vitro and in vivo studies. In biodistribution studies, the /sup 99m/Tc D-HF had a two-component blood clearance (half-times 1 hr and 15 hr) and was 80--88% coagulable. The /sup 99m/Tc AM-Fab retained its immunoreactivity as tested by affinity chromatography; also during in vivo localization in experimental myocardial infarction. This labeling technique provides an easy and efficient approach to the /sup 99m/Tc labeling of other biologically active and acid-labile macromolecules

  1. Allosteric communication in myosin V: from small conformational changes to large directed movements.

    Directory of Open Access Journals (Sweden)

    M Cecchini

    Full Text Available The rigor to post-rigor transition in myosin, a consequence of ATP binding, plays an essential role in the Lymn-Taylor functional cycle because it results in the dissociation of the actomyosin complex after the powerstroke. On the basis of the X-ray structures of myosin V, we have developed a new normal mode superposition model for the transition path between the two states. Rigid-body motions of the various subdomains and specific residues at the subdomain interfaces are key elements in the transition. The allosteric communication between the nucleotide binding site and the U50/L50 cleft is shown to result from local changes due to ATP binding, which induce large amplitude motions that are encoded in the structure of the protein. The triggering event is the change in the interaction of switch I and the P-loop, which is stabilized by ATP binding. The motion of switch I, which is a relatively rigid element of the U50 subdomain, leads directly to a partial opening of the U50/L50 cleft; the latter is expected to weaken the binding of myosin to actin. The calculated transition path demonstrates the nature of the subdomain coupling and offers an explanation for the mutual exclusion of ATP and actin binding. The mechanism of the uncoupling of the converter from the motor head, an essential part of the transition, is elucidated. The origin of the partial untwisting of the central beta-sheet in the rigor to post-rigor transition is described.

  2. Auxotonic to isometric contraction transitioning in a beating heart causes myosin step-size to down shift.

    Directory of Open Access Journals (Sweden)

    Thomas P Burghardt

    Full Text Available Myosin motors in cardiac ventriculum convert ATP free energy to the work of moving blood volume under pressure. The actin bound motor cyclically rotates its lever-arm/light-chain complex linking motor generated torque to the myosin filament backbone and translating actin against resisting force. Previous research showed that the unloaded in vitro motor is described with high precision by single molecule mechanical characteristics including unitary step-sizes of approximately 3, 5, and 8 nm and their relative step-frequencies of approximately 13, 50, and 37%. The 3 and 8 nm unitary step-sizes are dependent on myosin essential light chain (ELC N-terminus actin binding. Step-size and step-frequency quantitation specifies in vitro motor function including duty-ratio, power, and strain sensitivity metrics. In vivo, motors integrated into the muscle sarcomere form the more complex and hierarchically functioning muscle machine. The goal of the research reported here is to measure single myosin step-size and step-frequency in vivo to assess how tissue integration impacts motor function. A photoactivatable GFP tags the ventriculum myosin lever-arm/light-chain complex in the beating heart of a live zebrafish embryo. Detected single GFP emission reports time-resolved myosin lever-arm orientation interpreted as step-size and step-frequency providing single myosin mechanical characteristics over the active cycle. Following step-frequency of cardiac ventriculum myosin transitioning from low to high force in relaxed to auxotonic to isometric contraction phases indicates that the imposition of resisting force during contraction causes the motor to down-shift to the 3 nm step-size accounting for >80% of all the steps in the near-isometric phase. At peak force, the ATP initiated actomyosin dissociation is the predominant strain inhibited transition in the native myosin contraction cycle. The proposed model for motor down-shifting and strain sensing involves ELC N

  3. Myosin Binding Protein-C Slow Phosphorylation is Altered in Duchenne Dystrophy and Arthrogryposis Myopathy in Fast-Twitch Skeletal Muscles.

    Science.gov (United States)

    Ackermann, Maegen A; Ward, Christopher W; Gurnett, Christina; Kontrogianni-Konstantopoulos, Aikaterini

    2015-08-19

    Myosin Binding Protein-C slow (sMyBP-C), encoded by MYBPC1, comprises a family of regulatory proteins of skeletal muscles that are phosphorylated by PKA and PKC. MYBPC1 missense mutations are linked to the development of Distal Arthrogryposis-1 (DA-1). Although structure-function details for this myopathy are evolving, function is undoubtedly driven by sequence variations and post-translational modifications in sMyBP-C. Herein, we examined the phosphorylation profile of sMyBP-C in mouse and human fast-twitch skeletal muscles. We used Flexor Digitorum Brevis (FDB) isolated from young (~2-months old) and old (~14-months old) wild type and mdx mice, and human Abductor Hallucis (AH) and gastrocnemious muscles carrying the DA-1 mutations. Our results indicate both constitutive and differential phosphorylation of sMyBP-C in aged and diseased muscles. We report a 7-35% reduction in the phosphorylation levels of select sites in old wild type and young or old mdx FDB mouse muscles, compared to young wild type tissue. Similarly, we observe a 30-70% decrease in the phosphorylation levels of all PKA and PKC phospho-sites in the DA-1 AH, but not gastrocnemius, muscle. Overall, our studies show that the phosphorylation pattern of sMyBP-C is differentially regulated in response to age and disease, suggesting that phosphorylation plays important roles in these processes.

  4. Age- and Activity-Related Differences in the Abundance of Myosin Essential and Regulatory Light Chains in Human Muscle

    Directory of Open Access Journals (Sweden)

    James N. Cobley

    2016-04-01

    Full Text Available Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM. We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping.

  5. Harmonic Force Spectroscopy Reveals a Force-Velocity Curve from a Single Human Beta Cardiac Myosin Motor

    DEFF Research Database (Denmark)

    Sung, Jongmin; Nag, Suman; Vestergaard, Christian L.

    2014-01-01

    human beta cardiac myosin S1. We also compare load-velocity curves for wild-type motors with load-velocity curves of mutant forms that cause hypertrophic or dilated-cardiomyopathy (HCM or DCM), in order to understand the effects of mutations on the contractile cycle at the single molecule level....

  6. The structure of the actin-smooth muscle myosin motor domain complex in the rigor state.

    Science.gov (United States)

    Banerjee, Chaity; Hu, Zhongjun; Huang, Zhong; Warrington, J Anthony; Taylor, Dianne W; Trybus, Kathleen M; Lowey, Susan; Taylor, Kenneth A

    2017-12-01

    Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a ∼6 Å resolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4 Å) and lowest (∼6-7 Å) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin II bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle.

    Science.gov (United States)

    Cooke, Roger

    2011-03-01

    Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response to exposure to cold or to overfeeding. The identification of the "furnace" that is responsible for increased heat generation in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX), with a very slow ATP turnover rate has recently been observed in skeletal muscle (Stewart et al. in Proc Natl Acad Sci USA 107:430-435, 2010). Inhibition of the myosin ATPase activity in the SRX was suggested to be caused by binding of the myosin head to the core of the thick filament in a structural motif identified earlier by electron microscopy. To be compatible with the basal metabolic rate observed in vivo for resting muscle, most myosin heads would have to be in the SRX. Modulation of the population of this state, relative to the normal relaxed state, was proposed to be a major contributor to adaptive thermogenesis in resting muscle. Transfer of only 20% of myosin heads from the SRX into the normal relaxed state would cause muscle thermogenesis to double. Phosphorylation of the myosin regulatory light chain was shown to transfer myosin heads from the SRX into the relaxed state, which would increase thermogenesis. In particular, thermogenesis by myosin has been proposed to play a role in the dissipation of calories during overfeeding. Up-regulation of muscle thermogenesis by pharmaceuticals that target the SRX would provide new approaches to the treatment of obesity or high blood sugar levels.

  8. Structural insights into the globular tails of the human type v myosins Myo5a, Myo5b, And Myo5c.

    Directory of Open Access Journals (Sweden)

    Hana Velvarska

    Full Text Available Vertebrate type V myosins (MyoV Myo5a, Myo5b, and Myo5c mediate transport of several different cargoes. All MyoV paralogs bind to cargo complexes mainly by their C-terminal globular domains. In absence of cargo, the globular domain of Myo5a inhibits its motor domain. Here, we report low-resolution SAXS models for the globular domains from human Myo5a, Myo5b, and Myo5c, which suggest very similar overall shapes of all three paralogs. We determined the crystal structures of globular domains from Myo5a and Myo5b, and provide a homology model for human Myo5c. When we docked the Myo5a crystal structure into a previously published electron microscopy density of the autoinhibited full-length Myo5a, only one domain orientation resulted in a good fit. This structural arrangement suggests the participation of additional region of the globular domain in autoinhibition. Quantification of the interaction of the Myo5a globular domain with its motor complex revealed a tight binding with dissociation half-life in the order of minutes, suggesting a rather slow transition between the active and inactive states.

  9. Life without double-headed non-muscle myosin II motor proteins

    Science.gov (United States)

    Betapudi, Venkaiah

    2014-07-01

    Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC) belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients’ life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.

  10. Life without double-headed non-muscle myosin II motor proteins

    Directory of Open Access Journals (Sweden)

    Venkaiah eBetapudi

    2014-07-01

    Full Text Available Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients’ life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.

  11. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    Science.gov (United States)

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-02-12

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

  12. Structural characterization of the interactions between calmodulin and skeletal muscle myosin light chain kinase: Effect of peptide (576-594)G binding on the Ca2+-binding domains

    International Nuclear Information System (INIS)

    Seeholzer, S.H.; Wand, A.J.

    1989-01-01

    Calcium-containing calmodulin (CaM) and its complex with a peptide corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase [skMLCK(576-594)G] have been studied by one- and two-dimensional 1 H NMR techniques. Resonances arising from the antiparallel β-sheet structures associated with the calcium-binding domains of CaM and their counterparts in the CaM-skMLCK(576-594)G complex have been assigned. The assignments were initiated by application of the main chain directed assignment strategy. It is found that, despite significant changes in chemical shifts of resonances arising from amino acid residues in this region upon binding of the peptide, the β-sheets have virtually the same structure in the complex as in CaM. Hydrogen exchange rates of amide NH within the β-sheet structures are significantly slowed upon binding of peptide. These data, in conjunction with the observed nuclear Overhauser effect (NOE) patterns and relative intensities and the downfield shifts of associated amide and α resonances upon binding of peptide, show that the peptide stabilizes the Ca 2+ -bound state of calmodulin. The observed pattern of NOEs within the β-sheets and their structural similarity correspond closely to those predicted by the crystal structure. These findings imply that the apparent inconsistency of the crystal structure with recently reported low-angle X-ray scattering profiles of CaM may lie within the putative central helix bridging the globular domains

  13. The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses α Myosin Heavy-Chain Gene Expression

    Science.gov (United States)

    Sucharov, Carmen C.; Helmke, Steve M.; Langer, Stephen J.; Perryman, M. Benjamin; Bristow, Michael; Leinwand, Leslie

    2004-01-01

    Human heart failure is accompanied by repression of genes such as α myosin heavy chain (αMyHC) and SERCA2A and the induction of fetal genes such as βMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human αMyHC promoter. We have now identified a region of the αMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human αMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases αMyHC mRNA expression and increases skeletal α-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the αMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human αMyHC promoter during heart failure. PMID:15367688

  14. Pesquisa de marcadores para os genes da cadeia pesada da beta-miosina cardíaca e da proteína C de ligação à miosina em familiares de pacientes com cardiomiopatia hipertrófica Research of markers for the genes of the heavy chain of cardiac beta-myosin and myosin binding protein C in relatives of patients with hypertrophic cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Adriana Paula Tirone

    2005-06-01

    Full Text Available OBJETIVO: Estudar os marcadores moleculares para os genes da cadeia pesada da beta-miosina cardíaca e da proteína-C de ligação à miosina em familiares de portadores de cardiomiopatia hipertrófica. MÉTODOS: Foram estudadas 12 famílias que realizaram anamnese, exame físico, eletrocardiograma, ecocardiograma e coleta de sangue para o estudo genético através da reação em cadeia da polimerasse. RESULTADOS: Dos 227 familiares 25% eram acometidos, sendo 51% do sexo masculino com idade média de 35±19 (2 a 95 anos. A análise genética mostrou ligação com o gene da b-miosina cardíaca em uma família e, em outra, ligação com o gene da proteína C de ligação à miosina. Em cinco famílias foram excluídas ligações com os dois genes; em duas, a ligação com o gene da proteína C de ligação à miosina, porém para o gene da b-miosina os resultados foram inconclusivos; em duas famílias os resultados foram inconclusivos para os dois genes e em uma foi excluída ligação para o gene da b-miosina mas ficou inconclusivo para o gene da proteína C de ligação à miosina. CONCLUSÃO: Em nosso meio, talvez predominem outros genes que não aqueles descritos na literatura, ou que existam outras diferenças genéticas relacionadas com a origem de nossa população e/ou fatores ambientais.OBJECTIVE: To study the molecular markers for the genes of the heavy chain of cardiac beta-myosin and the myosin binding protein C in relatives of carriers of hypertrophic cardiomyopathy. METHODS: Twelve families who had anamnesis, physical exam, electrocardiogram, echocardiogram and blood collection for the genetic study through the chain reaction of polymerase. RESULTS: From the 227 relatives, 25% were ill-taken, with 51% men, with an average age of 35±19 (2 to 95 years old. The genetic analysis showed a connection with the gene of the cardiac b-myosin in a family and, in another, a connection with the gene of the myosin-binding protein C. In five

  15. Characterization of human cardiac myosin heavy chain genes

    International Nuclear Information System (INIS)

    Yamauchi-Takihara, K.; Sole, M.J.; Liew, J.; Ing, D.; Liew, C.C.

    1989-01-01

    The authors have isolated and analyzed the structure of the genes coding for the α and β forms of the human cardiac myosin heavy chain (MYHC). Detailed analysis of four overlapping MYHC genomic clones shows that the α-MYHC and β-MYHC genes constitute a total length of 51 kilobases and are tandemly linked. The β-MYHC-encoding gene, predominantly expressed in the normal human ventricle and also in slow-twitch skeletal muscle, is located 4.5 kilobases upstream of the α-MYHC-encoding gene, which is predominantly expressed in normal human atrium. The authors have determined the nucleotide sequences of the β form of the MYHC gene, which is 100% homologous to the cardiac MYHC cDNA clone (pHMC3). It is unlikely that the divergence of a few nucleotide sequences from the cardiac β-MYHC cDNA clone (pHMC3) reported in a MYHC cDNA clone (PSMHCZ) from skeletal muscle is due to a splicing mechanism. This finding suggests that the same β form of the cardiac MYHC gene is expressed in both ventricular and slow-twitch skeletal muscle. The promoter regions of both α- and β-MYHC genes, as well as the first four coding regions in the respective genes, have also been sequenced. The sequences in the 5'-flanking region of the α- and β-MYHC-encoding genes diverge extensively from one another, suggesting that expression of the α- and β-MYHC genes is independently regulated

  16. Cardiac Myosin Binding Protein-C Autoantibodies are Potential Early Indicators of Cardiac Dysfunction and Patient Outcome in Acute Coronary Syndrome.

    Science.gov (United States)

    Lynch, Thomas L; Kuster, Diederik W D; Gonzalez, Beverly; Balasubramanian, Neelam; Nair, Nandini; Day, Sharlene; Calvino, Jenna E; Tan, Yanli; Liebetrau, Christoph; Troidl, Christian; Hamm, Christian W; Güçlü, Ahmet; McDonough, Barbara; Marian, Ali J; van der Velden, Jolanda; Seidman, Christine E; Huggins, Gordon S; Sadayappan, Sakthivel

    2017-04-01

    The degradation and release of cardiac myosin binding protein-C (cMyBP-C) upon cardiac damage may stimulate an inflammatory response and autoantibody (AAb) production. We determined whether the presence of cMyBP-C-AAbs associated with adverse cardiac function in CVD patients. Importantly, cMyBP-C-AAbs were significantly detected in ACS patient sera upon arrival to the emergency department, particularly in STEMI patients. Patients positive for cMyBP-C-AAbs had a reduced LVEF and elevated levels of clinical biomarkers of MI. We conclude that cMyBP-C-AAbs may serve as early predictive indicators of deteriorating cardiac function and patient outcome in ACS patients prior to the infarction.

  17. Alternative S2 Hinge Regions of the Myosin Rod Affect Myofibrillar Structure and Myosin Kinetics

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Mark S.; Dambacher, Corey M.; Knowles, Aileen F.; Braddock, Joan M.; Farman, Gerrie P.; Irving, Thomas C.; Swank, Douglas M.; Bernstein, Sanford I.; Maughan, David W.; (RPI); (IIT); (SDSU); (Vermont)

    2009-07-01

    The subfragment 2/light meromyosin 'hinge' region has been proposed to significantly contribute to muscle contraction force and/or speed. Transgenic replacement of the endogenous fast muscle isovariant hinge A (exon 15a) in Drosophila melanogaster indirect flight muscle with the slow muscle hinge B (exon 15b) allows examination of the structural and functional changes when only this region of the myosin molecule is different. Hinge B was previously shown to increase myosin rod length, increase A-band and sarcomere length, and decrease flight performance compared to hinge A. We applied additional measures to these transgenic lines to further evaluate the consequences of modifying this hinge region. Structurally, the longer A-band and sarcomere lengths found in the hinge B myofibrils appear to be due to the longitudinal addition of myosin heads. Functionally, hinge B, although a significant distance from the myosin catalytic domain, alters myosin kinetics in a manner consistent with this region increasing myosin rod length. These structural and functional changes combine to decrease whole fly wing-beat frequency and flight performance. Our results indicate that this hinge region plays an important role in determining myosin kinetics and in regulating thick and thin filament lengths as well as sarcomere length.

  18. K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase

    International Nuclear Information System (INIS)

    Nakanishi, S.; Yamada, K.; Kase, H.; Nakamura, S.; Nonomura, Y.

    1988-01-01

    Effects of K-252a, purified from the culture broth of Nocardiopsis sp., on the activity of myosin (light chain kinase were investigated. 1) K-252a affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca 2+ -dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca 2+ -independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10 -6 M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10 -4 M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lowere in the presence of 100 μM ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using [γ- 32 P]ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP. These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase

  19. Clinical features and prognostic implications of familial hypertrophic cardiomyopathy related to the cardiac myosin-binding protein C gene.

    Science.gov (United States)

    Charron, P; Dubourg, O; Desnos, M; Bennaceur, M; Carrier, L; Camproux, A C; Isnard, R; Hagege, A; Langlard, J M; Bonne, G; Richard, P; Hainque, B; Bouhour, J B; Schwartz, K; Komajda, M

    1998-06-09

    Little information is available on phenotype-genotype correlations in familial hypertrophic cardiomyopathy that are related to the cardiac myosin binding protein C (MYBPC3) gene. The aim of this study was to perform this type of analysis. We studied 76 genetically affected subjects from nine families with seven recently identified mutations (SASint20, SDSint7, SDSint23, branch point int23, Glu542Gln, a deletion in exon 25, and a duplication/deletion in exon 33) in the MYBPC3 gene. Detailed clinical, ECG, and echocardiographic parameters were analyzed. An intergene analysis was performed by comparing the MYBPC3 group to seven mutations in the beta-myosin heavy-chain gene (beta-MHC) group (n=52). There was no significant phenotypic difference among the different mutations in the MYBPC3 gene. However, in the MYBPC3 group compared with the beta-MHC group, (1) prognosis was significantly better (P<0.0001), and no deaths occurred before the age of 40 years; (2) the age at onset of symptoms was delayed (41+/-19 versus 35+/-17 years, P<0.002); and (3) before 30 years of age, the phenotype was particularly mild because penetrance was low (41% versus 62%), maximal wall thicknesses lower (12+/-4 versus 16+/-7 mm, P<0.03), and abnormal T waves less frequent (9% versus 45%, P<0.02). These results are consistent with specific clinical features related to the MYBPC3 gene: onset of the disease appears delayed and the prognosis is better than that associated with the beta-MHC gene. These findings could be particularly important for the purpose of clinical management and genetic counseling in familial hypertrophic cardiomyopathy.

  20. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    Science.gov (United States)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  1. IMP metabolism in human skeletal muscle after exhaustive exercise

    DEFF Research Database (Denmark)

    Tullson, P. C.; Bangsbo, Jens; Hellsten, Ylva

    1995-01-01

    This study addressed whether AMP deaminase (AMPD)myosin binding occurs with deamination during intense exercise in humans and the extent of purine loss from muscle during the initial minutes of recovery. Male subjects performed cycle exercise (265 +/- 2 W for 4.39 +/- 0.04 min) to stimulate muscle...... inosine 5'-monophosphate (IMP) formation. After exercise, blood flow to one leg was occluded. Muscle biopsies (vastus lateralis) were taken before and 3.6 +/- 0.2 min after exercise from the occluded leg and 0.7 +/- 0.0, 1.1 +/- 0.0, and 2.9 +/- 0.1 min postexercise in the nonoccluded leg. Exercise...... activated AMPD; at exhaustion IMP was 3.5 +/- 0.4 mmol/kg dry muscle. Before exercise, 16.0 +/- 1.6% of AMPD cosedimented with the myosin fraction; the extent of AMPD:myosin binding was unchanged by exercise. Inosine content increased about threefold during exercise and twofold more during recovery; by 2...

  2. Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Chung, Le Thi Kim; Hosaka, Toshio; Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka; Teshigawara, Kiyoshi; Sakai, Tohru; Nakaya, Yutaka; Funaki, Makoto

    2010-01-01

    In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

  3. Head-head interactions of resting myosin crossbridges in intact frog skeletal muscles, revealed by synchrotron x-ray fiber diffraction.

    Directory of Open Access Journals (Sweden)

    Kanji Oshima

    Full Text Available The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation

  4. Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

    Science.gov (United States)

    Boëda, Batiste; El-Amraoui, Aziz; Bahloul, Amel; Goodyear, Richard; Daviet, Laurent; Blanchard, Stéphane; Perfettini, Isabelle; Fath, Karl R.; Shorte, Spencer; Reiners, Jan; Houdusse, Anne; Legrain, Pierre; Wolfrum, Uwe; Richardson, Guy; Petit, Christine

    2002-01-01

    Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia. PMID:12485990

  5. Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.

    Science.gov (United States)

    Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

    2012-01-01

    Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

  6. Changes in the structure of calmodulin induced by a peptide based on the calmodulin-binding domain of myosin light chain kinase

    International Nuclear Information System (INIS)

    Heidorn, D.B.; Seeger, P.A.; Rokop, S.E.; Blumenthal, D.K.; Means, A.R.; Crespi, H.; Trewhella, J.

    1989-01-01

    Small-angle X-ray and neutron scattering data were used to study the solution structure of calmodulin complexed with a synthetic peptide corresponding to residues 577-603 of rabbit skeletal muscle myosin light chain kinase. The X-ray data indicate that, in the presence of Ca 2+ , the calmodulin-peptide complex has a structure that is considerably more compact than uncomplexed calmodulin. The radius of gyration, R g , for the complex is approximately 20% smaller than that of uncomplexed Ca 2+ ·calmodulin, and the maximum dimension, d max , for the complex is also about 20% smaller. The peptide-induced conformational rearrangement of calmodulin is [Ca 2+ ] dependent. The length distribution function for the complex is more symmetric than that for uncomplexed Ca 2+ ·calmodulin, indicating that more of the mass is distributed toward the center of mass for the complex, compared with the dumbbell-shaped Ca 2+ ·calmodulin. The solvent contrast dependence of R g for neutron scattering indicates that the peptide is located more toward the center of the complex, while the calmodulin is located more peripherally, and that the centers of mass of the calmodulin and the peptide are not coincident. The scattering data support the hypothesis that the interconnecting helix region observed in the crystal structure for calmodulin is quite flexible in solution, allowing the two lobes of calmodulin to form close contacts on binding the peptide. This flexibility of the central helix may play a critical role in activating target enzymes such as myosin light chain kinase

  7. Robust mechanobiological behavior emerges in heterogeneous myosin systems

    Science.gov (United States)

    Egan, Paul F.; Moore, Jeffrey R.; Ehrlicher, Allen J.; Weitz, David A.; Schunn, Christian; Cagan, Jonathan; LeDuc, Philip

    2017-09-01

    Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors. The computational approach models variations in single myosin molecular structure, system organization, and force stimuli to predict system behavior for filament velocity, energy consumption, and robustness. Robustness is the range of forces where a filament is expected to have continuous velocity and depends on used myosin system energy. Myosin systems are shown to have highly nonlinear behavior across force conditions that may be exploited at a systems level by combining slow and fast myosin isoforms heterogeneously. Results suggest some heterogeneous systems have lower energy use near stall conditions and greater energy consumption when unloaded, therefore promoting robustness. These heterogeneous system capabilities are unique in comparison with homogenous systems and potentially advantageous for high performance bionanotechnologies. Findings open doors at the intersections of mechanics and biology, particularly for understanding and treating myosin-related diseases and developing approaches for motor molecule-based technologies.

  8. A global, myosin light chain kinase-dependent increase in myosin II contractility accompanies the metaphase-anaphase transition in sea urchin eggs.

    Science.gov (United States)

    Lucero, Amy; Stack, Christianna; Bresnick, Anne R; Shuster, Charles B

    2006-09-01

    Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase-anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase-anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus.

  9. A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase–Anaphase Transition in Sea Urchin Eggs

    Science.gov (United States)

    Lucero, Amy; Stack, Christianna; Bresnick, Anne R.

    2006-01-01

    Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase–anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase–anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus. PMID:16837551

  10. Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat.

    Science.gov (United States)

    Andruchov, Oleg; Galler, Stefan

    2008-03-01

    This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca(2+) activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.

  11. Coupling between myosin head conformation and the thick filament backbone structure.

    Science.gov (United States)

    Hu, Zhongjun; Taylor, Dianne W; Edwards, Robert J; Taylor, Kenneth A

    2017-12-01

    The recent high-resolution structure of the thick filament from Lethocerus asynchronous flight muscle shows aspects of thick filament structure never before revealed that may shed some light on how striated muscles function. The phenomenon of stretch activation underlies the function of asynchronous flight muscle. It is most highly developed in flight muscle, but is also observed in other striated muscles such as cardiac muscle. Although stretch activation is likely to be complex, involving more than a single structural aspect of striated muscle, the thick filament itself, would be a prime site for regulatory function because it must bear all of the tension produced by both its associated myosin motors and any externally applied force. Here we show the first structural evidence that the arrangement of myosin heads within the interacting heads motif is coupled to the structure of the thick filament backbone. We find that a change in helical angle of 0.16° disorders the blocked head preferentially within the Lethocerus interacting heads motif. This observation suggests a mechanism for how tension affects the dynamics of the myosin heads leading to a detailed hypothesis for stretch activation and shortening deactivation, in which the blocked head preferentially binds the thin filament followed by the free head when force production occurs. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Binding of corroded ions to human saliva.

    Science.gov (United States)

    Mueller, H J

    1985-05-01

    Employing equilibrium dialysis, the binding abilities of Cu, Al, Co and Cr ions from corroded Cu-Al and Co-Cr dental casting alloys towards human saliva and two of its gel chromatographic fractions were determined. Results indicate that both Cu and Co bind to human saliva i.e. 0.045 and 0.027 mg/mg protein, respectively. Besides possessing the largest binding ability, Cu also possessed the largest binding capacity. The saturation of Cu binding was not reached up to the limit of 0.35 mg protein/ml employed in the tests, while Co reached full saturation at about 0.2 mg protein/ml. Chromium showed absolutely no binding to human saliva while Al ions did not pass through the dialysis membranes. Compared to the binding with solutions that were synthetically made up to contain added salivary-type proteins, it is shown that the binding to human saliva is about 1 order of magnitude larger, at least for Cu ions.

  13. Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.

    Science.gov (United States)

    Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B Cicero; Zarzoso, Manuel; Ramirez, Rafael J; Sener, Michelle F; Mundada, Lakshmi V; Klos, Matthew; Devaney, Eric J; Vikstrom, Karen L; Herron, Todd J; Jalife, José

    2013-11-01

    Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. © 2013.

  14. Characteristics of myosin profile in human vastus lateralis muscle in relation to training background.

    Science.gov (United States)

    Zawadowska, B; Majerczak, J; Semik, D; Karasinski, J; Kolodziejski, L; Kilarski, W M; Duda, K; Zoladz, J A

    2004-01-01

    Twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg) of different training background were investigated and classified into three groups according to their physical activity and sport discipline: untrained students (group A), national and sub-national level endurance athletes (group B, 7.8+/-2.9 years of specialised training) and sprint-power athletes (group C, 12.8+/-8.7 years of specialised training). Muscle biopsies of vastus lateralis were analysed histochemically for mATPase and SDH activities, immunohistochemically for fast and slow myosin, and electrophoretically followed by Western immunoblotting for myosin heavy chain (MyHC) composition. Significant differences (Pski-jumping, volleyball, soccer and modern dance. Furthermore, the relative amount of the fastest MyHCIIX isoform in vastus lateralis muscle was significantly lower in the athletes from group C than in students (group A). We conclude that the myosin profile in the athletes belonging to group C was unfavourable for their sport disciplines. This could be the reason why those athletes did not reach international level despite of several years of training.

  15. The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability.

    Science.gov (United States)

    Smelter, Dan F; de Lange, Willem J; Cai, Wenxuan; Ge, Ying; Ralphe, J Carter

    2018-06-01

    Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of

  16. Characterization of the stimulation of human platelets by stable analogues of PGH2/TXA2

    International Nuclear Information System (INIS)

    Morinelli, T.A.

    1987-01-01

    The specific effects of the TXA 2 /PGH 2 analogues, U46619 (9,11-dideoxy,9α-11α-methanoepoxy-PGF/sub 2α/), and 9,11 aza-PGH 2 , on human platelet shape change, myosin light chain phosphorylation, serotonin release, fibrinogen receptor exposure and platelet aggregation were measured and compared with binding of 3 H-U46619 to platelets. Shape change and myosin light chain phosphorylation were found to saturable and dose dependent. These two effects were competitively inhibited by specific antagonists of TXA 2 /PGH 2 receptors (BM13177 and I-PTA-OH) indicating that they are receptor mediated. Binding of 3 H-U46619 showed two components. Occupancy of high affinity binding sites correlated with platelet shape change and myosin and light chain phosphorylation. A second component with an apparent K/sub d/ of 1.46 +/- 0.47 μM, may represent a second, low-affinity site. Therefore, the platelet release reaction as not directly correlated with occupancy of high affinity receptors but could be related to the second binding component of U46619. Fibrinogen receptor exposure and platelet aggregation caused by U46619 appeared to be events mediated by the release of ADP from platelet dense granules

  17. CHARACTERIZATION OF TIGHTLY-ASSOCIATED SMOOTH MUSCLE MYOSIN-MYOSIN LIGHT CHAIN KINASE-CALMODULIN COMPLEXES*

    OpenAIRE

    Hong, Feng; Haldeman, Brian D.; John, Olivia A.; Brewer, Paul D.; Wu, Yi-Ying; Ni, Shaowei; Wilson, David P.; Walsh, Michael P.; Baker, Jonathan E.; Cremo, Christine R.

    2009-01-01

    A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by smooth muscle myosin light chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM (up-SMM) from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73 ± 9), the ratio was ~ 23–37% of that in gizzard tissue. Fifteen t...

  18. Human chorionic ganodotropin binding sites in the human endometrium

    International Nuclear Information System (INIS)

    Bhattacharya, S.; Banerjee, J.; Sen, S.; Manna, P.R.

    1993-01-01

    The existence of high-affinity and low-capacity specific binding sites for luteinizing hormone/human chorionic gonadotropin (hCG) has been reported in porcine, rabbit and rat uteri. The authors have identified the hCG binding sites in the human endometrium collected from 35-42-year-old ovulatory and anovulatory women. The binding characteristics of hCG to endometrial tissue preparations from ovulatory and anovulatory women showed saturability with high affinity and low capacity. Scatchard plot analysis showed the dissociation constant of specific binding sites in the ovulatory women to be 3.5x10 -10 mol/l and in anovulatory women to be 3.1x10 -10 mol/l. The maximum binding capacity varied considerably between ovulatory and anovulatory endometrium. Among the divalent metal ions tested Zn 2+ effected a remarkable increase in [ 125 I]hCG binding to the endometrium, whereas Mn 2+ showed a marginal increase and other metal ions did not have any effect. Data obtained with human endometrium indicate an influence of the functional state of the ovary on [ 125 I]hCG binding to endometrium. 14 refs., 3 figs

  19. Endocytosis of Integrin-Binding Human Picornaviruses

    Directory of Open Access Journals (Sweden)

    Pirjo Merilahti

    2012-01-01

    Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  20. Myosin II dynamics are regulated by tension in intercalating cells.

    Science.gov (United States)

    Fernandez-Gonzalez, Rodrigo; Simoes, Sérgio de Matos; Röper, Jens-Christian; Eaton, Suzanne; Zallen, Jennifer A

    2009-11-01

    Axis elongation in Drosophila occurs through polarized cell rearrangements driven by actomyosin contractility. Myosin II promotes neighbor exchange through the contraction of single cell boundaries, while the contraction of myosin II structures spanning multiple pairs of cells leads to rosette formation. Here we show that multicellular actomyosin cables form at a higher frequency than expected by chance, indicating that cable assembly is an active process. Multicellular cables are sites of increased mechanical tension as measured by laser ablation. Fluorescence recovery after photobleaching experiments show that myosin II is stabilized at the cortex in regions of increased tension. Myosin II is recruited in response to an ectopic force and relieving tension leads to a rapid loss of myosin, indicating that tension is necessary and sufficient for cortical myosin localization. These results demonstrate that myosin II dynamics are regulated by tension in a positive feedback loop that leads to multicellular actomyosin cable formation and efficient tissue elongation.

  1. Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes.

    Directory of Open Access Journals (Sweden)

    Hector N Aguilar

    Full Text Available The 'phosphate-binding tag' (phos-tag reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC in cultured human uterine myocytes.We have evaluated and validated the concept that, when using an antibody (Ab against the total-protein, the sum of all phosphorylation states in a single lane represents a 'closed system' since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT and calpeptin (Calp induce RLC kinase (MLCK- and rho-kinase (ROK-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional 'loading' or 'reference' standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.

  2. Characterization of myosin light chain in shrimp hemocytic phagocytosis.

    Science.gov (United States)

    Han, Fang; Wang, Zhiyong; Wang, Xiaoqing

    2010-11-01

    Myosin light chain, a well-known cytoskeleton gene, regulates multiple processes that are involved in material transport, muscle shrink and cell division. However, its function in phagocytosis against invading pathogens in crustacean remains unknown. In this investigation, a myosin light chain gene was obtained from Marsupenaeus japonicus shrimp. The full-length cDNA of this gene was of 766 bp and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids. The myosin light chain protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified GST fusion protein. As revealed by immuno-electron microscopy, the myosin light chain protein was only expressed in the dark bands of muscle. In the present study, the myosin light chain gene was up-regulated in the WSSV-resistant shrimp as revealed by real-time PCR and western blot. And the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were remarkably increased in the WSSV-resistant shrimp, suggesting that the myosin light chain protein was essential in hemocytic phagocytosis. On the other hand, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the myosin light chain gene was silenced by sequence-specific siRNA. These findings suggested that myosin light chain protein was involved in the regulation of hemocytic phagocytosis of shrimp. Copyright 2010 Elsevier Ltd. All rights reserved.

  3. Imaging of myocardial infarction in dogs and humans using monoclonal antibodies specific for human myosin heavy chains

    International Nuclear Information System (INIS)

    Leger, J.; Chevalier, J.; Larue, C.; Gautier, P.; Planchenault, J.; Aumaitre, E.; Messner, P.; Puech, P.; Saccavini, J.C.; Pau, B.

    1991-01-01

    The use of three different monoclonal antibodies specific for human ventricular myosin heavy chains in the visualization of the location and extent of necrosis in dogs with experimental acute myocardial infarction and in humans is described. Using a classic immunohistochemical method or ex vivo analysis of heart slices in dogs with acute myocardial infarction subjected to intravenous injection of unlabeled antimyosin antibodies or antimyosin antibodies labeled with indium-111, it was observed that all antibody fragments specifically reached the targeted necrotic zone less than 2 h after antibody injection and remained bound for up to 24 h. In a limited but significant number of cases (5 of the 12 humans and 11 of 43 dogs), it was possible to image the necrotic zone in vivo as early as 2 to 4 h after antibody injection. In other cases, individual blood clearance variations retarded or even prevented in vivo necrosis detection. Higher antimyosin fixation values were obtained in the necrotic zones in dogs with a rapid blood clearance relative to that of the other dogs. It is concluded that antimyosin antibodies always reached necrotic areas within 2 h. If blood clearance was rapid, in vivo imaging of the necrotic area was possible 2 to 6 h after necrosis, even in humans. In some cases, however, uncontrolled individual variations in the timing required for sufficient blood clearance hampered this rapid in vivo detection of myocardial necrosis

  4. Characteristics of myosin profile in human vastus lateralis muscle in relation to training background.

    Directory of Open Access Journals (Sweden)

    J A Zoladz

    2004-10-01

    Full Text Available Twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg of different training background were investigated and classified into three groups according to their physical activity and sport discipline: untrained students (group A, national and sub-national level endurance athletes (group B, 7.8+/-2.9 years of specialised training and sprint-power athletes (group C, 12.8+/-8.7 years of specialised training. Muscle biopsies of vastus lateralis were analysed histochemically for mATPase and SDH activities, immunohistochemically for fast and slow myosin, and electrophoretically followed by Western immunoblotting for myosin heavy chain (MyHC composition. Significant differences (P<0.05 regarding composition of muscle fibre types and myosin heavy chains were found only between groups A (41.7+/-1.6% of MyHCI, 40.8+/-4.0% of MyHCIIA and 17.5+/-4.0% of MyHCIIX and B (64.3+/-0.8% of MyHCI, 34.0+/-1.4% of MyHCIIA and 1.7+/-1.4% of MyHCIIX and groups A and C (59.6+/-1.6% of MyHCI, 37.2+/-1.3% of MyHCIIA and 3.2+/-1.3% of MyHCIIX. Unexpectedly, endurance athletes (group B such as long-distance runners, cyclists and cross country skiers, did not differ from the athletes representing short term, high power output sports (group C such as ice hockey, karate, ski-jumping, volleyball, soccer and modern dance. Furthermore, the relative amount of the fastest MyHCIIX isoform in vastus lateralis muscle was significantly lower in the athletes from group C than in students (group A. We conclude that the myosin profile in the athletes belonging to group C was unfavourable for their sport disciplines. This could be the reason why those athletes did not reach international level despite of several years of training.

  5. Reciprocal and dynamic polarization of planar cell polarity core components and myosin

    Science.gov (United States)

    Newman-Smith, Erin; Kourakis, Matthew J; Reeves, Wendy; Veeman, Michael; Smith, William C

    2015-01-01

    The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization. DOI: http://dx.doi.org/10.7554/eLife.05361.001 PMID:25866928

  6. Conformational distributions and proximity relationships in the rigor complex of actin and myosin subfragment-1.

    Science.gov (United States)

    Nyitrai, M; Hild, G; Lukács, A; Bódis, E; Somogyi, B

    2000-01-28

    Cyclic conformational changes in the myosin head are considered essential for muscle contraction. We hereby show that the extension of the fluorescence resonance energy transfer method described originally by Taylor et al. (Taylor, D. L., Reidler, J., Spudich, J. A., and Stryer, L. (1981) J. Cell Biol. 89, 362-367) allows determination of the position of a labeled point outside the actin filament in supramolecular complexes and also characterization of the conformational heterogeneity of an actin-binding protein while considering donor-acceptor distance distributions. Using this method we analyzed proximity relationships between two labeled points of S1 and the actin filament in the acto-S1 rigor complex. The donor (N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate) was attached to either the catalytic domain (Cys-707) or the essential light chain (Cys-177) of S1, whereas the acceptor (5-(iodoacetamido)fluorescein) was attached to the actin filament (Cys-374). In contrast to the narrow positional distribution (assumed as being Gaussian) of Cys-707 (5 +/- 3 A), the positional distribution of Cys-177 was found to be broad (102 +/- 4 A). Such a broad positional distribution of the label on the essential light chain of S1 may be important in accommodating the helically arranged acto-myosin binding relative to the filament axis.

  7. Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus

    Czech Academy of Sciences Publication Activity Database

    Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, V.; Rülicke, T.; Rathkolb, B.; Hans, W.; Bohla, A.; Eickelberg, O.; Stoeger, T.; Wolf, E.; Yildirim, A.Ö.; Gailus-Durner, V.; Fuchs, H.; de Angelis, M.H.; Hozák, Pavel

    2013-01-01

    Roč. 8, č. 4 (2013), e61406 E-ISSN 1932-6203 R&D Projects: GA ČR GAP305/11/2232; GA TA ČR TE01020022; GA MŠk LH12143; GA ČR(CZ) GD204/09/H084 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : nuclear myosin * myosin isoforms * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.534, year: 2013

  8. SwissProt search result: AK101919 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101919 J033071J10 (O14974) Protein phosphatase 1 regulatory subunit 12A (Myosin phosphatase target...ing subunit 1) (Myosin phosphatase target subunit 1) (Protein phosphatase myosin-binding subunit) MYPT1_HUMAN 1e-12 ...

  9. SwissProt search result: AK102584 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102584 J033098E05 (O14974) Protein phosphatase 1 regulatory subunit 12A (Myosin phosphatase target...ing subunit 1) (Myosin phosphatase target subunit 1) (Protein phosphatase myosin-binding subunit) MYPT1_HUMAN 6e-11 ...

  10. SwissProt search result: AK101292 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101292 J033033G18 (O14974) Protein phosphatase 1 regulatory subunit 12A (Myosin phosphatase target...ing subunit 1) (Myosin phosphatase target subunit 1) (Protein phosphatase myosin-binding subunit) MYPT1_HUMAN 1e-12 ...

  11. SwissProt search result: AK111573 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111573 J013073E13 (O14974) Protein phosphatase 1 regulatory subunit 12A (Myosin phosphatase target...ing subunit 1) (Myosin phosphatase target subunit 1) (Protein phosphatase myosin-binding subunit) MYPT1_HUMAN 3e-15 ...

  12. Localization of gonadotropin binding sites in human ovarian neoplasms

    International Nuclear Information System (INIS)

    Nakano, R.; Kitayama, S.; Yamoto, M.; Shima, K.; Ooshima, A.

    1989-01-01

    The binding of human luteinizing hormone and human follicle-stimulating hormone to ovarian tumor biopsy specimens from 29 patients was analyzed. The binding sites for human luteinizing hormone were demonstrated in one tumor of epithelial origin (mucinous cystadenoma) and in one of sex cord-stromal origin (theca cell tumor). The binding sites for human follicle-stimulating hormone were found in three tumors of epithelial origin (serous cystadenoma and mucinous cystadenoma) and in two of sex cord-stromal origin (theca cell tumor and theca-granulosa cell tumor). The surface-binding autoradiographic study revealed that the binding sites for gonadotropins were localized in the stromal tissue. The results suggest that gonadotropic hormones may play a role in the growth and differentiation of a certain type of human ovarian neoplasms

  13. A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects.

    Science.gov (United States)

    Suggs, Jennifer A; Melkani, Girish C; Glasheen, Bernadette M; Detor, Mia M; Melkani, Anju; Marsan, Nathan P; Swank, Douglas M; Bernstein, Sanford I

    2017-06-01

    Individuals with inclusion body myopathy type 3 (IBM3) display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K) in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in decreased in vitro

  14. A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects

    Directory of Open Access Journals (Sweden)

    Jennifer A. Suggs

    2017-06-01

    Full Text Available Individuals with inclusion body myopathy type 3 (IBM3 display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in

  15. Myosin heavy chain expression in rabbit masseter muscle during postnatal development

    NARCIS (Netherlands)

    Bredman, J. J.; Weijs, W. A.; Korfage, H. A.; Brugman, P.; Moorman, A. F.

    1992-01-01

    The expression of isoforms of myosin heavy chain (MHC) during postnatal development was studied in the masseter muscle of the rabbit. Evidence is presented that in addition to adult fast and slow myosin, the rabbit masseter contains neonatal and 'cardiac' alpha-MHC. During postnatal growth myosin

  16. Interaction of c-Cbl with myosin IIA regulates Bleb associated macropinocytosis of Kaposi's sarcoma-associated herpesvirus.

    Directory of Open Access Journals (Sweden)

    Mohanan Valiya Veettil

    2010-12-01

    Full Text Available KSHV is etiologically associated with Kaposi's sarcoma (KS, an angioproliferative endothelial cell malignancy. Macropinocytosis is the predominant mode of in vitro entry of KSHV into its natural target cells, human dermal microvascular endothelial (HMVEC-d cells. Although macropinocytosis is known to be a major route of entry for many viruses, the molecule(s involved in the recruitment and integration of signaling early during macropinosome formation is less well studied. Here we demonstrate that tyrosine phosphorylation of the adaptor protein c-Cbl is required for KSHV induced membrane blebbing and macropinocytosis. KSHV induced the tyrosine phosphorylation of c-Cbl as early as 1 min post-infection and was recruited to the sites of bleb formation. Infection also led to an increase in the interaction of c-Cbl with PI3-K p85 in a time dependent manner. c-Cbl shRNA decreased the formation of KSHV induced membrane blebs and macropinocytosis as well as virus entry. Immunoprecipitation of c-Cbl followed by mass spectrometry identified the interaction of c-Cbl with a novel molecular partner, non-muscle myosin heavy chain IIA (myosin IIA, in bleb associated macropinocytosis. Phosphorylated c-Cbl colocalized with phospho-myosin light chain II in the interior of blebs of infected cells and this interaction was abolished by c-Cbl shRNA. Studies with the myosin II inhibitor blebbistatin demonstrated that myosin IIA is a biologically significant component of the c-Cbl signaling pathway and c-Cbl plays a new role in the recruitment of myosin IIA to the blebs during KSHV infection. Myosin II associates with actin in KSHV induced blebs and the absence of actin and myosin ubiquitination in c-Cbl ShRNA cells suggested that c-Cbl is also responsible for the ubiquitination of these proteins in the infected cells. This is the first study demonstrating the role of c-Cbl in viral entry as well as macropinocytosis, and provides the evidence that a signaling complex

  17. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC

    Energy Technology Data Exchange (ETDEWEB)

    Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.; Domaradzki, Tera [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States); Hofmann, Wilma A., E-mail: whofmann@buffalo.edu [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States)

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.

  18. Myosin VIIa as a common component of cilia and microvilli.

    Science.gov (United States)

    Wolfrum, U; Liu, X; Schmitt, A; Udovichenko, I P; Williams, D S

    1998-01-01

    The distribution of myosin VIIa, which is defective or absent in Usher syndrome 1B, was studied in a variety of tissues by immunomicroscopy. The primary aim was to determine whether this putative actin-based mechanoenzyme is a common component of cilia. Previously, it has been proposed that defective ciliary function might be the basis of some forms of Usher syndrome. Myosin VIIa was detected in cilia from cochlear hair cells, olfactory neurons, kidney distal tubules, and lung bronchi. It was also found to cofractionate with the axonemal fraction of retinal photoreceptor cells. Immunolabeling appeared most concentrated in the periphery of the transition zone of the cilia. This general presence of a myosin in cilia is surprising, given that cilia are dominated by microtubules, and not actin filaments. In addition to cilia, myosin VIIa was also found in actin-rich microvilli of different types of cell. We conclude that myosin VIIa is a common component of cilia and microvilli.

  19. Mutation profile of all 49 exons of the human myosin VIIA gene, and haplotype analysis, in Usher 1B families from diverse origins.

    Science.gov (United States)

    Adato, A; Weil, D; Kalinski, H; Pel-Or, Y; Ayadi, H; Petit, C; Korostishevsky, M; Bonne-Tamir, B

    1997-10-01

    Usher syndrome types I (USH1A-USH1E) are a group of autosomal recessive diseases characterized by profound congenital hearing loss, vestibular areflexia, and progressive visual loss due to retinitis pigmentosa. The human myosin VIIA gene, located on 11q14, has been shown to be responsible for Usher syndrome type 1B (USH1B). Haplotypes were constructed in 28 USH1 families by use of the following polymorphic markers spanning the USH1B locus: D11S787, D11S527, D11S1789, D11S906, D11S4186, and OMP. Affected individuals and members of their families from 12 different ethnic origins were screened for the presence of mutations in all 49 exons of the myosin VIIA gene. In 15 families myosin VIIA mutations were detected, verifying their classification as USH1B. All these mutations are novel, including three missense mutations, one premature stop codon, two splicing mutations, one frameshift, and one deletion of >2 kb comprising exons 47 and 48, a part of exon 49, and the introns between them. Three mutations were shared by more than one family, consistent with haplotype similarities. Altogether, 16 USH1B haplotypes were observed in the 15 families; most haplotypes were population specific. Several exonic and intronic polymorphisms were also detected. None of the 20 known USH1B mutations reported so far in other world populations were identified in our families.

  20. Fast and slow myosins as markers of muscle injury.

    Science.gov (United States)

    Guerrero, M; Guiu-Comadevall, M; Cadefau, J A; Parra, J; Balius, R; Estruch, A; Rodas, G; Bedini, J L; Cussó, R

    2008-07-01

    The diagnosis of muscular lesions suffered by athletes is usually made by clinical criteria combined with imaging of the lesion (ultrasonography and/or magnetic resonance) and blood tests to detect the presence of non-specific muscle markers. This study was undertaken to evaluate injury to fast and slow-twitch fibres using specific muscle markers for these fibres. Blood samples were obtained from 51 non-sports people and 38 sportsmen with skeletal muscle injury. Western blood analysis was performed to determine fast and slow myosin and creatine kinase (CK) levels. Skeletal muscle damage was diagnosed by physical examination, ultrasonography and magnetic resonance and biochemical markers. The imaging tests were found to be excellent for detecting and confirming grade II and III lesions. However, grade I lesions were often unconfirmed by these techniques. Grade I lesions have higher levels of fast myosin than slow myosin with a very small increase in CK levels. Grade II and III lesions have high values of both fast and slow myosin. The evaluation of fast and slow myosin in the blood 48 h after the lesion occurs is a useful aid for the detection of type I lesions in particular, since fast myosin is an exclusive skeletal muscle marker. The correct diagnosis of grade I lesions can prevent progression of the injury in athletes undergoing continual training sessions and competitions, thus aiding sports physicians in their decision making.

  1. Biased Brownian motion mechanism for processivity and directionality of single-headed myosin-VI.

    Science.gov (United States)

    Iwaki, Mitsuhiro; Iwane, Atsuko Hikikoshi; Ikebe, Mitsuo; Yanagida, Toshio

    2008-01-01

    Conventional form to function as a vesicle transporter is not a 'single molecule' but a coordinated 'two molecules'. The coordinated two molecules make it complicated to reveal its mechanism. To overcome the difficulty, we adopted a single-headed myosin-VI as a model protein. Myosin-VI is an intracellular vesicle and organelle transporter that moves along actin filaments in a direction opposite to most other known myosin classes. The myosin-VI was expected to form a dimer to move processively along actin filaments with a hand-over-hand mechanism like other myosin organelle transporters. However, wild-type myosin-VI was demonstrated to be monomer and single-headed, casting doubt on its processivity. Using single molecule techniques, we show that green fluorescent protein (GFP)-fused single-headed myosin-VI does not move processively. However, when coupled to a 200 nm polystyrene bead (comparable to an intracellular vesicle in size) at a ratio of one head per bead, single-headed myosin-VI moves processively with large (40 nm) steps. Furthermore, we found that a single-headed myosin-VI-bead complex moved more processively in a high-viscous solution (40-fold higher than water) similar to cellular environment. Because diffusion of the bead is 60-fold slower than myosin-VI heads alone in water, we propose a model in which the bead acts as a diffusional anchor for the myosin-VI, enhancing the head's rebinding following detachment and supporting processive movement of the bead-monomer complex. This investigation will help us understand how molecular motors utilize Brownian motion in cells.

  2. The Kinetics of Myosin Light Chain Kinase Activation of Smooth Muscle Myosin in an In Vitro Model System

    OpenAIRE

    Hong, Feng; Facemyer, Kevin C.; Carter, Michael S.; Jackson, Del R.; Haldeman, Brian D.; Ruana, Nick; Sutherland, Cindy; Walsh, Michael P.; Cremo, Christine R.; Baker, Josh E.

    2013-01-01

    During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca2+-CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vi...

  3. Engineering controllable bidirectional molecular motors based on myosin

    Science.gov (United States)

    Chen, Lu; Nakamura, Muneaki; Schindler, Tony D.; Parker, David; Bryant, Zev

    2012-04-01

    Cytoskeletal motors drive the transport of organelles and molecular cargoes within cells and have potential applications in molecular detection and diagnostic devices. Engineering molecular motors with controllable properties will allow selective perturbation of mechanical processes in living cells and provide optimized device components for tasks such as molecular sorting and directed assembly. Biological motors have previously been modified by introducing activation/deactivation switches that respond to metal ions and other signals. Here, we show that myosin motors can be engineered to reversibly change their direction of motion in response to a calcium signal. Building on previous protein engineering studies and guided by a structural model for the redirected power stroke of myosin VI, we have constructed bidirectional myosins through the rigid recombination of structural modules. The performance of the motors was confirmed using gliding filament assays and single fluorophore tracking. Our strategy, in which external signals trigger changes in the geometry and mechanics of myosin lever arms, should make it possible to achieve spatiotemporal control over a range of motor properties including processivity, stride size and branchpoint turning.

  4. Mu opioid receptor binding sites in human brain

    International Nuclear Information System (INIS)

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand [ 3 H]DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of [ 3 H]DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas

  5. Dynamics of myosin II organization into cortical contractile networks and fibers

    Science.gov (United States)

    Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel; Jedlicka, Sabrina; Vavylonis, Dimitrios

    2014-03-01

    The morphology of adhered cells critically depends on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin which disrupts actomyosin stress fibers. Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared them to studies by other groups. This analysis suggested that the following processes contribute to the assembly of cortical actomyosin into fibers: random myosin mini-filament assembly and disassembly along the cortex; myosin mini-filament aligning and contraction; stabilization of cortical myosin upon increasing contractile tension. We developed simple numerical simulations that include those processes. The results of simulations of cells at steady state and in response to blebbistatin capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

  6. Unconstrained steps of myosin VI appear longest among known molecular motors.

    Science.gov (United States)

    Ali, M Yusuf; Homma, Kazuaki; Iwane, Atsuko Hikikoshi; Adachi, Kengo; Itoh, Hiroyasu; Kinosita, Kazuhiko; Yanagida, Toshio; Ikebe, Mitsuo

    2004-06-01

    Myosin VI is a two-headed molecular motor that moves along an actin filament in the direction opposite to most other myosins. Previously, a single myosin VI molecule has been shown to proceed with steps that are large compared to its neck size: either it walks by somehow extending its neck or one head slides along actin for a long distance before the other head lands. To inquire into these and other possible mechanism of motility, we suspended an actin filament between two plastic beads, and let a single myosin VI molecule carrying a bead duplex move along the actin. This configuration, unlike previous studies, allows unconstrained rotation of myosin VI around the right-handed double helix of actin. Myosin VI moved almost straight or as a right-handed spiral with a pitch of several micrometers, indicating that the molecule walks with strides slightly longer than the actin helical repeat of 36 nm. The large steps without much rotation suggest kinesin-type walking with extended and flexible necks, but how to move forward with flexible necks, even under a backward load, is not clear. As an answer, we propose that a conformational change in the lifted head would facilitate landing on a forward, rather than backward, site. This mechanism may underlie stepping of all two-headed molecular motors including kinesin and myosin V.

  7. Myosin-1C uses a novel phosphoinositide-dependent pathway for nuclear localization.

    Science.gov (United States)

    Nevzorov, Ilja; Sidorenko, Ekaterina; Wang, Weihuan; Zhao, Hongxia; Vartiainen, Maria K

    2018-02-01

    Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin-dependent motor protein Myosin-1C (Myo1C) resembles the diffusion-retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  8. Flexibility of the myosin heavy chain: direct evidence that the region containing SH/sub 1/ and SH/sub 2/ can move 10 /Angstrom/ under the influence of nucleotide binding

    Energy Technology Data Exchange (ETDEWEB)

    Huston, E.E.; Grammer, J.C.; Yount, R.G.

    1988-12-13

    Previous experiments demonstrated that two thiols of skeletal myosin subfragment 1 (SF/sub 1/) could be oxidized to a disulfide bond by treatment with a 2-fold excess of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) in the presence of MgADP. The resulting characteristic changes in the ATPase activities of SF/sub 1/ and the fact that MgADP was stably trapped at the active site, suggested that the two thiols cross-linked were SH/sub 1/ (Cys-707) and SH/sub 2/ (Cys-697) from the myosin heavy chain. To verify this suggestion, SF/sub 1/, after DTNB treatment as above, was treated with an excess of N-ethylmaleimide to block all accessible thiols. The single protein disulfide produced by DTNB oxidation was reduced with dithioerythritol and the modified SF/sub 1/ internally cross-linked with equimolar (/sup 14/C)p-phenylenedimaleimide (pPDM) in the presence of MgADP. After extensive trypsinization, the major /sup 14/C-labeled peptide was isolated, characterized, and shown to be Cys-Asn-Gly-Val-Leu-Gly-Ile-Arg-Ile-Cys-Arg, in which the two cysteines were cross-linked by pPDM. This peptide is known to contain SH/sub 2/ and SH/sub 1/ in this order and to come from residues 697-708 in the rabbit skeletal myosin heavy chain. Parallel experiments with (/sup 14/C)pPDM and unmodified SF/sub 1/ similar to those above gave an identical SH/sub 1/, SH/sub 2/ tryptic peptide, verifying earlier labeling results. These combined results demonstrate that SH/sub 1/ and SH/sub 2/ cross-linked by pPDM (12-13 /Angstrom/, S to S) or by oxidation with DTNB (2 /Angstrom/, S to S) can move a minimum of 10 /Angstrom/ under the influence of nucleotide binding. Because these residues are separated by only nine amino acids in the primary sequence, this small section of the heavy chain must possess extraordinary flexibility.

  9. Myosin light chain kinase phosphorylation in tracheal smooth muscle

    International Nuclear Information System (INIS)

    Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E.

    1990-01-01

    Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32 P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+

  10. Actin-myosin network is required for proper assembly of influenza virus particles

    Energy Technology Data Exchange (ETDEWEB)

    Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

    2015-02-15

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.

  11. Actin-myosin network is required for proper assembly of influenza virus particles

    International Nuclear Information System (INIS)

    Kumakura, Michiko; Kawaguchi, Atsushi; Nagata, Kyosuke

    2015-01-01

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network

  12. Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis.

    Directory of Open Access Journals (Sweden)

    Juliane Bremer

    2016-11-01

    Full Text Available During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, we show that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. We find that this alignment requires myosin phosphatase activity within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, we demonstrate that spinal motoneurons fine-tune their position during axonogenesis and we identify the myosin II regulatory network as a key regulator.

  13. Meningococcal factor H-binding protein vaccines with decreased binding to human complement factor H have enhanced immunogenicity in human factor H transgenic mice.

    Science.gov (United States)

    Rossi, Raffaella; Granoff, Dan M; Beernink, Peter T

    2013-11-04

    Factor H-binding protein (fHbp) is a component of a meningococcal vaccine recently licensed in Europe for prevention of serogroup B disease, and a second vaccine in clinical development. The protein specifically binds human factor H (fH), which down-regulates complement activation and enhances resistance to bactericidal activity. There are conflicting data from studies in human fH transgenic mice on whether binding of human fH to fHbp vaccines decreases immunogenicity, and whether mutant fHbp vaccines with decreased fH binding have enhanced immunogenicity. fHbp can be classified into two sub-families based on sequence divergence and immunologic cross-reactivity. Previous studies of mutant fHbp vaccines with low fH binding were from sub-family B, which account for approximately 60% of serogroup B case isolates. In the present study, we evaluated the immunogenicity of two mutant sub-family A fHbp vaccines containing single substitutions, T221A or D211A, which resulted in 15- or 30-fold lower affinity for human fH, respectively, than the corresponding control wild-type fHbp vaccine. In transgenic mice with high serum concentrations of human fH, both mutant vaccines elicited significantly higher IgG titers and higher serum bactericidal antibody responses than the control fHbp vaccine that bound human fH. Thus, mutations introduced into a sub-family A fHbp antigen to decrease fH binding can increase protective antibody responses in human fH transgenic mice. Collectively the data suggest that mutant fHbp antigens with decreased fH binding will result in superior vaccines in humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Engineering controllable bidirectional molecular motors based on myosin

    Science.gov (United States)

    Chen, Lu; Nakamura, Muneaki; Schindler, Tony D.; Parker, David; Bryant, Zev

    2012-01-01

    Cytoskeletal motors drive the transport of organelles and molecular cargoes within cells1, and have potential applications in molecular detection and diagnostic devices2,3. Engineering molecular motors with dynamically controllable properties will allow selective perturbation of mechanical processes in living cells, and yield optimized device components for complex tasks such as molecular sorting and directed assembly3. Biological motors have previously been modified by introducing activation/deactivation switches that respond to metal ions4,5 and other signals6. Here we show that myosin motors can be engineered to reversibly change their direction of motion in response to a calcium signal. Building on previous protein engineering studies7–11 and guided by a structural model12 for the redirected power stroke of myosin VI, we constructed bidirectional myosins through the rigid recombination of structural modules. The performance of the motors was confirmed using gliding filament assays and single fluorophore tracking. Our general strategy, in which external signals trigger changes in the geometry and mechanics of myosin lever arms, should enable spatiotemporal control over a range of motor properties including processivity, stride size13, and branchpoint turning14. PMID:22343382

  15. Myosin VIII regulates protonemal patterning and developmental timing in the moss Physcomitrella patens.

    Science.gov (United States)

    Wu, Shu-Zon; Ritchie, Julie A; Pan, Ai-Hong; Quatrano, Ralph S; Bezanilla, Magdalena

    2011-09-01

    Plants have two classes of myosins. While recent work has focused on class XI myosins showing that myosin XI is responsible for organelle motility and cytoplasmic streaming, much less is known about the role of myosin VIII in plant growth and development. We have used a combination of RNAi and insertional knockouts to probe myosin VIII function in the moss Physcomitrella patens. We isolated Δmyo8ABCDE plants demonstrating that myosin VIII is not required for plant viability. However, myosin VIII mutants are smaller than wild-type plants in part due to a defect in cell size. Additionally, Δmyo8ABCDE plants produce more side branches and form gametophores much earlier than wild-type plants. In the absence of nutrient media, Δmyo8ABCDE plants exhibit significant protonemal patterning defects, including highly curved protonemal filaments, morphologically defective side branches, as well as an increase in the number of branches. Exogenous auxin partially rescues protonemal defects in Δmyo8ABCDE plants grown in the absence of nutrients. This result, together with defects in protonemal branching, smaller caulonemal cells, and accelerated development in the Δmyo8ABCDE plants, suggests that myosin VIII is involved in hormone homeostasis in P. patens.

  16. The expression of myosin genes in developing skeletal muscle in the mouse embryo

    International Nuclear Information System (INIS)

    Lyons, G.E.; Ontell, M.; Cox, R.; Sassoon, D.; Buckingham, M.

    1990-01-01

    Using in situ hybridization, we have investigated the temporal sequence of myosin gene expression in the developing skeletal muscle masses of mouse embryos. The probes used were isoform-specific, 35S-labeled antisense cRNAs to the known sarcomeric myosin heavy chain and myosin alkali light chain gene transcripts. Results showed that both cardiac and skeletal myosin heavy chain and myosin light chain mRNAs were first detected between 9 and 10 d post coitum (p.c.) in the myotomes of the most rostral somites. Myosin transcripts appeared in more caudal somites at later stages in a developmental gradient. The earliest myosin heavy chain transcripts detected code for the embryonic skeletal (MHCemb) and beta-cardiac (MHC beta) isoforms. Perinatal myosin heavy chain (MHCpn) transcripts begin to accumulate at 10.5 d p.c., which is much earlier than previously reported. At this stage, MHCemb is the major MHC transcript. By 12.5 d p.c., MHCpn and MHCemb mRNAs are present to an equal extent, and by 15.5 d p.c. the MHCpn transcript is the major MHC mRNA detected. Cardiac MHC beta transcripts are always present as a minor component. In contrast, the cardiac MLC1A mRNA is initially more abundant than that encoding the skeletal MLC1F isoform. By 12.5 d p.c. the two MLC mRNAs are present at similar levels, and by 15.5 d p.c., MLC1F is the predominant MLC transcript detected. Transcripts for the ventricular/slow (MLC1V) and another fast skeletal myosin light chain (MLC3F) are not detected in skeletal muscle before 15 d p.c., which marks the beginning of the fetal stage of muscle development. This is the first stage at which we can detect differences in expression of myosin genes between developing muscle fibers. We conclude that, during the development of the myotome and body wall muscles, different myosin genes follow independent patterns of activation and acculumation

  17. Nonmuscle myosin IIA and IIB differentially contribute to intrinsic and directed migration of human embryonic lung fibroblasts.

    Science.gov (United States)

    Kuragano, Masahiro; Murakami, Yota; Takahashi, Masayuki

    2018-03-25

    Nonmuscle myosin II (NMII) plays an essential role in directional cell migration. In this study, we investigated the roles of NMII isoforms (NMIIA and NMIIB) in the migration of human embryonic lung fibroblasts, which exhibit directionally persistent migration in an intrinsic manner. NMIIA-knockdown (KD) cells migrated unsteadily, but their direction of migration was approximately maintained. By contrast, NMIIB-KD cells occasionally reversed their direction of migration. Lamellipodium-like protrusions formed in the posterior region of NMIIB-KD cells prior to reversal of the migration direction. Moreover, NMIIB KD led to elongation of the posterior region in migrating cells, probably due to the lack of load-bearing stress fibers in this area. These results suggest that NMIIA plays a role in steering migration by maintaining stable protrusions in the anterior region, whereas NMIIB plays a role in maintenance of front-rear polarity by preventing aberrant protrusion formation in the posterior region. These distinct functions of NMIIA and NMIIB might promote intrinsic and directed migration of normal human fibroblasts. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod

    Energy Technology Data Exchange (ETDEWEB)

    Andreas, Michael P.; Ajay, Gautam; Gellings, Jaclyn A.; Rayment, Ivan (UW)

    2017-12-01

    X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 – Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.

  19. Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay

    International Nuclear Information System (INIS)

    Katus, H.A.; Hurrell, J.G.; Matsueda, G.R.; Ehrlich, P.; Zurawski, V.R. Jr.; Khaw, B.-A.; Haber, E.

    1982-01-01

    An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to differential between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (lC5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125 I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, lC5 and 2B9 were 25% cross reactive together, lC5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive. (author)

  20. Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links

    DEFF Research Database (Denmark)

    Lametsch, Marianne Lund; Luxford, Catherine; Skibsted, Leif Horsfelt

    2008-01-01

    of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation...

  1. Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

    Science.gov (United States)

    Oshiro, Satoshi; Honda, Shinya

    2014-04-18

    Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

  2. (TH) diazepam binding to human granulocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bond, P.A.; Cundall, R.L.; Rolfe, B.

    1985-07-08

    (TH)-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These (TH)-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14 M). Binding of (TH) diazepam at 0 is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109 +/- 17f moles per mg of protein and K/sub D/ of 3.07 +/- 0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 +/- 0.014. Binding is time dependent and reversible and it varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. 11 references, 3 figures, 1 table.

  3. Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II

    Directory of Open Access Journals (Sweden)

    Drescher Uwe

    2010-06-01

    Full Text Available Abstract Background In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A. It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear. Results In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK and myosin light chain kinase (MLCK, which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points. Conclusions Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.

  4. Adult fast myosin pattern and Ca2+-induced slow myosin pattern in primary skeletal muscle culture

    Science.gov (United States)

    Kubis, Hans-Peter; Haller, Ernst-August; Wetzel, Petra; Gros, Gerolf

    1997-01-01

    A primary muscle cell culture derived from newborn rabbit muscle and growing on microcarriers in suspension was established. When cultured for several weeks, the myotubes in this model develop the completely adult pattern of fast myosin light and heavy chains. When Ca2+ ionophore is added to the culture medium on day 11, raising intracellular [Ca2+] about 10-fold, the myotubes develop to exhibit properties of an adult slow muscle by day 30, expressing slow myosin light as well as heavy chains, elevated citrate synthase, and reduced lactate dehydrogenase. The remarkable plasticity of these myotubes becomes apparent, when 8 days after withdrawal of the ionophore a marked slow-to-fast transition, as judged from the expression of isomyosins and metabolic enzymes, occurs. PMID:9108130

  5. Catalytic strategy used by the myosin motor to hydrolyze ATP.

    Science.gov (United States)

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-07-22

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum-classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO4(2-) state, but in the H2PγO4(-) state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3(-)) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.

  6. Specific binding of beta-endorphin to normal human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chenet, B.; Hollis, V. Jr.; Kang, Y.; Simpkins, C.

    1986-03-05

    Beta-endorphin (BE) exhibits peripheral functions which may not be mediated by interactions with receptors in the brain. Recent studies have demonstrated binding of BE to both opioid and non-opioid receptors on lymphocytes and monocytes. Abood has reported specific binding of /sup 3/H-dihydromorphine in erythrocytes. Using 5 x 10/sup -11/M /sup 125/I-beta-endorphin and 10/sup -5/M unlabeled BE, they have detected 50% specific binding to human erythrocytes. This finding is supported by results from immunoelectron microscopy using rabbit anti-BE antibody and biotinylated secondary antibody with avidin-biotin complexes horseradish peroxidase. Binding is clearly observed and is confined to only one side of the cells. Conclusions: (1) BE binding to human erythrocytes was demonstrated by radioreceptor assay and immunoelectron microscopy, and (2) BE binding sites exist on only one side of the cells.

  7. Extracellular matrix-dependent myosin dynamics during G1-S phase cell cycle progression in hepatocytes

    International Nuclear Information System (INIS)

    Bhadriraju, Kiran; Hansen, Linda K.

    2004-01-01

    Cell spreading and proliferation are tightly coupled in anchorage-dependent cells. While adhesion-dependent proliferation signals require an intact actin cytoskeleton, and some of these signals such as ERK activation have been characterized, the role of myosin in spreading and cell cycle progression under different extracellular matrix (ECM) conditions is not known. Studies presented here examine changes in myosin activity in freshly isolated hepatocytes under ECM conditions that promote either proliferation (high fibronectin density) or growth arrest (low fibronectin density). Three different measures were obtained and related to both spreading and cell cycle progression: myosin protein levels and association with cytoskeleton, myosin light chain phosphorylation, and its ATPase activity. During the first 48 h in culture, corresponding with transit through G1 phase, there was a six-fold increase in both myosin protein levels and myosin association with actin cytoskeleton. There was also a steady increase in myosin light chain phosphorylation and ATPase activity with spreading, which did not occur in non-spread, growth-arrested cells on low density of fibronectin. Myosin-inhibiting drugs blocked ERK activation, cyclin D1 expression, and S phase entry. Overexpression of the cell cycle protein cyclin D1 overcame both ECM-dependent and actomyosin-dependent inhibition of DNA synthesis, suggesting that cyclin D1 is a key event downstream of myosin-dependent cell cycle regulation

  8. A Collapsin Response Mediator Protein 2 Isoform Controls Myosin II-Mediated Cell Migration and Matrix Assembly by Trapping ROCK II

    Science.gov (United States)

    Morgan-Fisher, Marie; Wait, Robin; Couchman, John R.; Wewer, Ulla M.

    2012-01-01

    Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells. PMID:22431514

  9. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

    Ahmed, M.S.; Zhou, D.H.; Maulik, D.; Eldefrawi, M.E.

    1990-01-01

    [ 3 H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [ 3 H]-cocaine with a high affinity site of 170 fmole/mg protein. The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [ 3 H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S 20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear

  10. Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility.

    Science.gov (United States)

    Green, Judith L; Wall, Richard J; Vahokoski, Juha; Yusuf, Noor A; Ridzuan, Mohd A Mohd; Stanway, Rebecca R; Stock, Jessica; Knuepfer, Ellen; Brady, Declan; Martin, Stephen R; Howell, Steven A; Pires, Isa P; Moon, Robert W; Molloy, Justin E; Kursula, Inari; Tewari, Rita; Holder, Anthony A

    2017-10-27

    Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain-interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Localization of Myosin and Actin in the Pelage and Whisker Hair Follicles of Rat

    International Nuclear Information System (INIS)

    Morioka, Kiyokazu; Matsuzaki, Toshiyuki; Takata, Kuniaki

    2006-01-01

    The combined effects of myosin II and actin enable muscle and nonmuscle cells to generate forces required for muscle contraction, cell division, cell migration, cellular morphological changes, the maintenance of cellular tension and polarity, and so on. However, except for the case of muscle contraction, the details are poorly understood. We focus on nonmuscle myosin and actin in the formation and maintenance of hair and skin, which include highly active processes in mammalian life with respect to the cellular proliferation, differentiation, and movement. The localization of nonmuscle myosin II and actin in neonatal rat dorsal skin, mystacial pad, hair follicles, and vibrissal follicles was studied by immunohistochemical technique to provide the basis for the elucidation of the roles of these proteins. Specificities of the antibodies were verified by using samples from the relevant tissues and subjecting them to immunoblotting test prior to morphological analyses. The myosin and actin were abundant and colocalized in the spinous and granular layers but scarce in the basal layer of the dorsal and mystacial epidermis. In hair and vibrissal follicles, nonmuscle myosin and actin were colocalized in the outer root sheath and some hair matrix cells adjoining dermal papillae. In contrast, most areas of the inner root sheath and hair matrix appeared to comprise very small amounts of myosin and actin. Hair shaft may comprise significant myosin during the course of its keratinization. These results suggest that the actin-myosin system plays a part in cell movement, differentiation, protection and other key functions of skin and hair cells

  12. Reversible covalent binding of neratinib to human serum albumin in vitro.

    Science.gov (United States)

    Chandrasekaran, Appavu; Shen, Li; Lockhead, Susan; Oganesian, Aram; Wang, Jianyao; Scatina, JoAnn

    2010-12-01

    Neratinib (HKI-272), an irreversible inhibitor of Her 2 tyrosine kinase, is currently in development as an alternative for first and second line therapy in metastatic breast cancer patients who overexpress Her 2. Following incubation of [(14)C]neratinib in control human plasma at 37°C for 6 hours, about 60% to 70% of the radioactivity was not extractable, due to covalent binding to albumin. In this study, factors that could potentially affect the covalent binding of neratinib to plasma proteins, specifically to albumin were investigated. When [(14)C]neratinib was incubated at 10 μg/mL in human serum albumin (HSA) or control human plasma, the percent binding increased with time; the highest percentages of binding (46 and 67%, respectively) were observed at 6 hours, the longest duration of incubation examined. Binding increased with increasing temperature; the highest percentages of binding to HSA or human plasma (59 and 78%) were observed at 45°C, the highest temperature tested. The binding also increased with increasing pH of incubation; the highest percentages of binding (56 and 65%) were observed at pH 8.5, the highest pH value tested. The percentages of binding were similar (53% to 57%) when a wide range of concentrations of [(14)C]neratinib (50 ng/mL to 10 μg/mL) were incubated with human plasma at 37°C for 6 hours, indicating that the binding was independent of the substrate concentration, especially in the therapeutic range (50 to 200 ng/mL). When human plasma proteins containing covalently bound [(14)C]neratinb were suspended in a 10 fold volume of phosphate buffer at pH 4.0, 6.0, 7.4, and 8.5, and further incubated at 37°C for ~ 16 hours, about 45%, 44%, 32%, and 12% of the total radioactivity, respectively, was released as unchanged [(14)C]neratinib, indicating that the binding is reversible in nature, with more released at pH 7.4 and below. In conclusion, the covalent binding of neratinib to serum albumin is pH, time and temperature dependent, but

  13. Reduction in beta-myosin heavy chains in stunned myocardium as assessed by nondenaturing gel electrophoresis.

    Science.gov (United States)

    Garcia, S C; Pomblum, V J; Gams, E; Rupp, H; Schipke, J D

    2007-09-01

    Myosin plays a key role in the structure and function of cardiac muscle. Three myosin isoenzymes (V(1), V(2), and V(3)) with different ATPase activities have been identified in mammalian ventricles based on their heavy chain constituents. The relative amount of myosin isoenzymes changes under physiological and pathological conditions. Until now, myosin isoenzymes have frequently been determined using either tube gel (nondenaturing) polyacrylamide gel electrophoresis (PAGE), or gradient or uniform sodium dodecyl sulfate (denaturing) PAGE. Both methods have disadvantages, e.g., a long running time. We developed, therefore, a uniform, nondenaturing PAGE with slab minigel format for analyzing the myosin isoenzymes in normoxic and stunned rabbit hearts. In normoxic hearts of adult rabbits, V(3) predominated over V(1) (46 vs 41%). In turn, in the stunned hearts, V(1) predominated over V(3) (70 vs 30%), and the heterodimeric V(2) was not anymore detectable. This alteration appears to result from a selective loss of myosin heavy chain (MHC)-beta. In parallel, the biochemical markers troponin I and creatine kinase were increased in the stunned hearts. We suggest that alterations of myosin isoenzymes in stunned myocardium can be monitored with native PAGE. The present analysis of myosin isoenzyme appears thus as a new tool for evaluating defects in MHC dimer formation in postischemic hearts.

  14. Binding of (/sup 3/H)imipramine to human platelet membranes with compensation for saturable binding to filters and its implication for binding studies with brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, O.M.; Wood, K.M.; Williams, D.C.

    1984-08-01

    Apparent specific binding of (/sup 3/H)imipramine to human platelet membranes at high concentrations of imipramine showed deviation from that expected of a single binding site, a result consistent with a low-affinity binding site. The deviation was due to displaceable, saturable binding to the glass fibre filters used in the assays. Imipramine, chloripramine, desipramine, and fluoxetine inhibited binding to filters whereas 5-hydroxytryptamine and ethanol were ineffective. Experimental conditions were developed that eliminated filter binding, allowing assay of high- and low-affinity binding to membranes. Failure to correct for filter binding may lead to overestimation of binding parameters, Bmax and KD for high-affinity binding to membranes, and may also be misinterpreted as indicating a low-affinity binding component in both platelet and brain membranes. Low-affinity binding (KD less than 2 microM) of imipramine to human platelet membranes was demonstrated and its significance discussed.

  15. Inhibitors of serotonin reuptake and specific imipramine binding in human blood plasma

    International Nuclear Information System (INIS)

    Brusov, O.S.; Fomenko, A.M.; Katasonov, A.B.; Lidemann, R.R.

    1985-01-01

    This paper describes a method of extraction of endogenous inhibitors of specific IMI binding and of 5-HT reuptake, from human blood plasma and the heterogeneity of these compounds is demonstrated. Specific binding was determined as the difference between binding of 3 H-IMI in the absence and in the presence of 50 microM IMI. Under these conditions, specific binding amounted to 70-80% of total binding of 3 H-IMI. It is shown that extract obtained from human blood contains a material which inhibits dose-dependently both 5-HT reuptake and specific binding of 3 H-IMI. Gel-chromatography of extracts of human blood plasma on Biogel P-2 is also shown

  16. An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells.

    Science.gov (United States)

    Yokota, Etsuo; Ueda, Shunpei; Tamura, Kentaro; Orii, Hidefumi; Uchi, Satoko; Sonobe, Seiji; Hara-Nishimura, Ikuko; Shimmen, Teruo

    2009-01-01

    The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175 kDa heavy chain (175 kDa myosin) and the other of 170 kDa heavy chain (170 kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175 kDa and 170 kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175 kDa myosin heavy chain showed that the 175 kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175 kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175 kDa myosin. Furthermore, this myosin was co-fractionated with GFP-ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175 kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells.

  17. Effects of exercise on insulin binding to human muscle

    International Nuclear Information System (INIS)

    Bonen, A.; Tan, M.H.; Clune, P.; Kirby, R.L.

    1985-01-01

    A procedure was developed to measure insulin binding to human skeletal muscle obtained via the percutaneous muscle biopsy technique. With this method the effects of exercise on insulin binding were investigated. Subjects (n = 9) exercised for 60 min on a bicycle ergometer at intensities ranging from 20-86% maximum O 2 consumption (VO 2 max). Blood samples were obtained before, during, and after exercise and analyzed for glucose and insulin. Muscle samples (250 mg) for the vastus lateralis were obtained 30 min before exercise, at the end of exercise, and 60 min after exercise. Two subjects rested during the experimental period. There was no linear relationship between exercise intensities and the changes in insulin binding to human muscle. At rest (n = 2) and at exercise intensities below 60% VO 2 max (n = 5) no change in insulin binding occurred (P greater than 0.05). However, when exercise occurred at greater than or equal to 69% VO 2 max (n = 4), a pronounced decrement in insulin binding (30-50%) was observed (P less than 0.05). This persisted for 60 min after exercise. These results indicate that insulin binding in human muscle is not altered by 60 min of exercise at less than or equal to 60% VO 2 max but that a marked decrement occurs when exercise is greater than or equal to 69% VO 2 max

  18. Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators for the treatment of systolic heart failure.

    Science.gov (United States)

    Manickam, Manoj; Jalani, Hitesh B; Pillaiyar, Thanigaimalai; Sharma, Niti; Boggu, Pulla Reddy; Venkateswararao, Eeda; Lee, You-Jung; Jeon, Eun-Seok; Jung, Sang-Hun

    2017-07-07

    A series of flexible urea derivatives have been synthesized and demonstrated as selective cardiac myosin ATPase activator. Among them 1-phenethyl-3-(3-phenylpropyl)urea (1, cardiac myosin ATPase activation at 10 μM = 51.1%; FS = 18.90; EF = 12.15) and 1-benzyl-3-(3-phenylpropyl)urea (9, cardiac myosin ATPase activation = 53.3%; FS = 30.04; EF = 18.27) showed significant activity in vitro and in vivo. The change of phenyl ring with tetrahydropyran-4-yl moiety viz., 1-(3-phenylpropyl)-3-((tetrahydro-2H-pyran-4-yl)methyl)urea (14, cardiac myosin ATPase activation = 81.4%; FS = 20.50; EF = 13.10), and morpholine moiety viz., 1-(2-morpholinoethyl)-3-(3-phenylpropyl)urea (21, cardiac myosin ATPase activation = 44.0%; FS = 24.79; EF = 15.65), proved to be efficient to activate the cardiac myosin. The potent compounds 1, 9, 14 and 21 were found to be selective for cardiac myosin over skeletal and smooth myosins. Thus, these urea derivatives are potent scaffold to develop as a newer cardiac myosin activator for the treatment of systolic heart failure. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  19. Cortical mechanics and myosin-II abnormalities associated with post-ovulatory aging: implications for functional defects in aged eggs

    Science.gov (United States)

    Mackenzie, Amelia C.L.; Kyle, Diane D.; McGinnis, Lauren A.; Lee, Hyo J.; Aldana, Nathalia; Robinson, Douglas N.; Evans, Janice P.

    2016-01-01

    STUDY HYPOTHESIS Cellular aging of the egg following ovulation, also known as post-ovulatory aging, is associated with aberrant cortical mechanics and actomyosin cytoskeleton functions. STUDY FINDING Post-ovulatory aging is associated with dysfunction of non-muscle myosin-II, and pharmacologically induced myosin-II dysfunction produces some of the same deficiencies observed in aged eggs. WHAT IS KNOWN ALREADY Reproductive success is reduced with delayed fertilization and when copulation or insemination occurs at increased times after ovulation. Post-ovulatory aged eggs have several abnormalities in the plasma membrane and cortex, including reduced egg membrane receptivity to sperm, aberrant sperm-induced cortical remodeling and formation of fertilization cones at the site of sperm entry, and reduced ability to establish a membrane block to prevent polyspermic fertilization. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Ovulated mouse eggs were collected at 21–22 h post-human chorionic gonadotrophin (hCG) (aged eggs) or at 13–14 h post-hCG (young eggs), or young eggs were treated with the myosin light chain kinase (MLCK) inhibitor ML-7, to test the hypothesis that disruption of myosin-II function could mimic some of the effects of post-ovulatory aging. Eggs were subjected to various analyses. Cytoskeletal proteins in eggs and parthenogenesis were assessed using fluorescence microscopy, with further analysis of cytoskeletal proteins in immunoblotting experiments. Cortical tension was measured through micropipette aspiration assays. Egg membrane receptivity to sperm was assessed in in vitro fertilization (IVF) assays. Membrane topography was examined by low-vacuum scanning electron microscopy (SEM). MAIN RESULTS AND THE ROLE OF CHANCE Aged eggs have decreased levels and abnormal localizations of phosphorylated myosin-II regulatory light chain (pMRLC; P = 0.0062). Cortical tension, which is mediated in part by myosin-II, is reduced in aged mouse eggs when compared with

  20. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...

  1. Thrombin binding to human brain and spinal cord

    International Nuclear Information System (INIS)

    McKinney, M.; Snider, R.M.; Richelson, E.

    1983-01-01

    Thrombin, a serine protease that regulates hemostasis, has been shown to stimulate the formation of cGMP in murine neuroblastoma cells. The nervous system in vivo thus may be postulated to respond to this blood-borne factor after it breaches the blood-brain barrier, as in trauma. Human alpha-thrombin was radiolabeled with 125I and shown to bind rapidly, reversibly, and with high affinity to human brain and spinal cord. These findings indicate the presence of specific thrombin-binding sites in nervous tissue and may have important clinical implications

  2. Dynamics of myosin II organization into contractile networks and fibers at the medial cell cortex

    Science.gov (United States)

    Nie, Wei

    The cellular morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of non-muscle myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin (which disrupts actomyosin stress fibers). Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared to studies by other groups. This analysis suggested the following processes: myosin minifilament assembly and disassembly; aligning and contraction; myosin filament stabilization upon increasing contractile tension. Numerical simulations that include those processes capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. We discuss methods to monitor myosin reorganization using non-linear imaging methods.

  3. Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

    Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

  4. Mechanochemical coupling in the myosin motor domain. I. Insights from equilibrium active-site simulations.

    Directory of Open Access Journals (Sweden)

    Haibo Yu

    2007-02-01

    Full Text Available Although the major structural transitions in molecular motors are often argued to couple to the binding of Adenosine triphosphate (ATP, the recovery stroke in the conventional myosin has been shown to be dependent on the hydrolysis of ATP. To obtain a clearer mechanistic picture for such "mechanochemical coupling" in myosin, equilibrium active-site simulations with explicit solvent have been carried out to probe the behavior of the motor domain as functions of the nucleotide chemical state and conformation of the converter/relay helix. In conjunction with previous studies of ATP hydrolysis with different active-site conformations and normal mode analysis of structural flexibility, the results help establish an energetics-based framework for understanding the mechanochemical coupling. It is proposed that the activation of hydrolysis does not require the rotation of the lever arm per se, but the two processes are tightly coordinated because both strongly couple to the open/close transition of the active site. The underlying picture involves shifts in the dominant population of different structural motifs as a consequence of changes elsewhere in the motor domain. The contribution of this work and the accompanying paper [] is to propose the actual mechanism behind these "population shifts" and residues that play important roles in the process. It is suggested that structural flexibilities at both the small and large scales inherent to the motor domain make it possible to implement tight couplings between different structural motifs while maintaining small free-energy drops for processes that occur in the detached states, which is likely a feature shared among many molecular motors. The significantly different flexibility of the active site in different X-ray structures with variable level arm orientations supports the notation that external force sensed by the lever arm may transmit into the active site and influence the chemical steps (nucleotide

  5. Human Masseter Muscle Fibers From the Elderly Express Less Neonatal Myosin Than Those of Young Adults

    Czech Academy of Sciences Publication Activity Database

    Cvetko, E.; Karen, Petr; Janáček, Jiří; Kubínová, Lucie; Plasencia, A.L.; Eržen, I.

    2012-01-01

    Roč. 295, č. 8 (2012), s. 1364-1372 ISSN 1932-8486 R&D Projects: GA MŠk(CZ) LC06063; GA MŠk(CZ) MEB090910 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : aging * confocal microscopy * myosin heavy chain * immunohistochemistry * muscle fiber types Subject RIV: FH - Neurology Impact factor: 1.343, year: 2012

  6. Native myosin from adult rabbit skeletal muscle: isoenzymes and states of aggregation.

    Science.gov (United States)

    Morel, J E; D'hahan, N; Taouil, K; Francin, M; Aguilar, A; Dalbiez, J P; Merah, Z; Grussaute, H; Hilbert, B; Ollagnon, F; Selva, G; Piot, F

    1998-04-21

    The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb).

  7. Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle.

    Science.gov (United States)

    Copeland, O'Neal; Sadayappan, Sakthivel; Messer, Andrew E; Steinen, Ger J M; van der Velden, Jolanda; Marston, Steven B

    2010-12-01

    A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n=21). Total phosphorylation was 2.7 ± 0.07 mol Pi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n=19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the

  8. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  9. Myosin light chain kinase regulates synaptic plasticity and fear learning in the lateral amygdala.

    Science.gov (United States)

    Lamprecht, R; Margulies, D S; Farb, C R; Hou, M; Johnson, L R; LeDoux, J E

    2006-01-01

    Learning and memory depend on signaling molecules that affect synaptic efficacy. The cytoskeleton has been implicated in regulating synaptic transmission but its role in learning and memory is poorly understood. Fear learning depends on plasticity in the lateral nucleus of the amygdala. We therefore examined whether the cytoskeletal-regulatory protein, myosin light chain kinase, might contribute to fear learning in the rat lateral amygdala. Microinjection of ML-7, a specific inhibitor of myosin light chain kinase, into the lateral nucleus of the amygdala before fear conditioning, but not immediately afterward, enhanced both short-term memory and long-term memory, suggesting that myosin light chain kinase is involved specifically in memory acquisition rather than in posttraining consolidation of memory. Myosin light chain kinase inhibitor had no effect on memory retrieval. Furthermore, ML-7 had no effect on behavior when the training stimuli were presented in a non-associative manner. Anatomical studies showed that myosin light chain kinase is present in cells throughout lateral nucleus of the amygdala and is localized to dendritic shafts and spines that are postsynaptic to the projections from the auditory thalamus to lateral nucleus of the amygdala, a pathway specifically implicated in fear learning. Inhibition of myosin light chain kinase enhanced long-term potentiation, a physiological model of learning, in the auditory thalamic pathway to the lateral nucleus of the amygdala. When ML-7 was applied without associative tetanic stimulation it had no effect on synaptic responses in lateral nucleus of the amygdala. Thus, myosin light chain kinase activity in lateral nucleus of the amygdala appears to normally suppress synaptic plasticity in the circuits underlying fear learning, suggesting that myosin light chain kinase may help prevent the acquisition of irrelevant fears. Impairment of this mechanism could contribute to pathological fear learning.

  10. Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

    Science.gov (United States)

    Kim, Kyoungtae; Keller, Thomas C S

    2002-01-07

    Smooth muscle cells use an actin-myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

  11. Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cells

    Science.gov (United States)

    Lehmann, Maik J.; Sherer, Nathan M.; Marks, Carolyn B.; Pypaert, Marc; Mothes, Walther

    2005-01-01

    Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, “surfing” toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body. PMID:16027225

  12. Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cells.

    Science.gov (United States)

    Lehmann, Maik J; Sherer, Nathan M; Marks, Carolyn B; Pypaert, Marc; Mothes, Walther

    2005-07-18

    Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, "surfing" toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.

  13. FR-like EBNA1 binding repeats in the human genome

    International Nuclear Information System (INIS)

    D'Herouel, Aymeric Fouquier; Birgersdotter, Anna; Werner, Maria

    2010-01-01

    Epstein-Barr virus (EBV) is widely spread in the human population. EBV nuclear antigen 1 (EBNA1) is a transcription factor that activates viral genes and is necessary for viral replication and partitioning, which binds the EBV genome cooperatively. We identify similar EBNA1 repeat binding sites in the human genome using a nearest-neighbor positional weight matrix. Previously experimentally verified EBNA1 sites in the human genome are successfully recovered by our approach. Most importantly, 40 novel regions are identified in the human genome, constituted of tandemly repeated binding sites for EBNA1. Genes located in the vicinity of these regions are presented as possible targets for EBNA1-mediated regulation. Among these, four are discussed in more detail: IQCB1, IMPG1, IRF2BP2 and TPO. Incorporating the cooperative actions of EBNA1 is essential when identifying regulatory regions in the human genome and we believe the findings presented here are highly valuable for the understanding of EBV-induced phenotypic changes.

  14. Increased myosin heavy chain-beta with atrial expression of ventricular light chain-2 in canine cardiomyopathy.

    Science.gov (United States)

    Fuller, Geraldine A; Bicer, Sabahattin; Hamlin, Robert L; Yamaguchi, Mamoru; Reiser, Peter J

    2007-10-01

    Dilated cardiomyopathy is a naturally occurring disease in humans and dogs. Human studies have shown increased levels of myosin heavy chain (MHC)-beta in failing ventricles and the left atria (LA) and of ventricular light chain (VLC)-2 in the right atria in dilated cardiomyopathy. This study evaluates the levels of MHC-beta in all heart chambers in prolonged canine right ventricular pacing. In addition, we determined whether levels of VLC2 were altered in these hearts. Failing hearts demonstrated significantly increased levels of MHC-beta in the right atria, right atrial appendage, LA, left atrial appendage (LAA), and right ventricle compared with controls. Significant levels of VLC2 were detected in the right atria of paced hearts. Differences in MHC-beta expression were observed between the LA and the LAA of paced and control dogs. MHC-beta expression was significantly greater in the LA of paced and control dogs compared with their respective LAA. The cardiac myosin isoform shifts in this study were similar to those observed in end-stage human heart failure and more severe than those reported in less prolonged pacing models, supporting the use of this model for further study of end-stage human heart failure. The observation of consistent differences between sampling sites, especially LA versus LAA, indicates the need for rigorous sampling consistency in future studies.

  15. Aspirin and salicylate bind to immunoglobulin heavy chain binding protein (BiP) and inhibit its ATPase activity in human fibroblasts.

    Science.gov (United States)

    Deng, W G; Ruan, K H; Du, M; Saunders, M A; Wu, K K

    2001-11-01

    Salicylic acid (SA), an endogenous signaling molecule of plants, possesses anti-inflammatory and anti-neoplastic actions in human. Its derivative, aspirin, is the most commonly used anti-inflammatory and analgesic drug. Aspirin and sodium salicylate (salicylates) have been reported to have multiple pharmacological actions. However, it is unclear whether they bind to a cellular protein. Here, we report for the first time the purification from human fibroblasts of a approximately 78 kDa salicylate binding protein with sequence identity to immunoglobulin heavy chain binding protein (BiP). The Kd values of SA binding to crude extract and to recombinant BiP were 45.2 and 54.6 microM, respectively. BiP is a chaperone protein containing a polypeptide binding site recognizing specific heptapeptide sequence and an ATP binding site. A heptapeptide with the specific sequence displaced SA binding in a concentration-dependent manner whereas a control heptapeptide did not. Salicylates inhibited ATPase activity stimulated by this specific heptapeptide but did not block ATP binding or induce BiP expression. These results indicate that salicylates bind specifically to the polypeptide binding site of BiP in human cells that may interfere with folding and transport of proteins important in inflammation.

  16. Electron Microscopic Recording of the Power and Recovery Strokes of Individual Myosin Heads Coupled with ATP Hydrolysis: Facts and Implications

    Directory of Open Access Journals (Sweden)

    Haruo Sugi

    2018-05-01

    Full Text Available The most straightforward way to get information on the performance of individual myosin heads producing muscle contraction may be to record their movement, coupled with ATP hydrolysis, electron-microscopically using the gas environmental chamber (EC. The EC enables us to visualize and record ATP-induced myosin head movement in hydrated skeletal muscle myosin filaments. When actin filaments are absent, myosin heads fluctuate around a definite neutral position, so that their time-averaged mean position remains unchanged. On application of ATP, myosin heads are found to move away from, but not towards, the bare region, indicating that myosin heads perform a recovery stroke (average amplitude, 6 nm. After exhaustion of ATP, myosin heads return to their neutral position. In the actin–myosin filament mixture, myosin heads form rigor actin myosin linkages, and on application of ATP, they perform a power stroke by stretching adjacent elastic structures because of a limited amount of applied ATP ≤ 10 µM. The average amplitude of the power stroke is 3.3 nm and 2.5 nm at the distal and the proximal regions of the myosin head catalytic domain (CAD, respectively. The power stroke amplitude increases appreciably at low ionic strength, which is known to enhance Ca2+-activated force in muscle. In both the power and recovery strokes, myosin heads return to their neutral position after exhaustion of ATP.

  17. [Myosin B ATPase activity of the intestinal smooth muscle in intestinal obstruction].

    Science.gov (United States)

    Takamatsu, H

    1983-06-01

    Intestinal smooth myosin B was prepared from muscle layers around the lesion in dogs with experimental colonic stenosis and in patients with congenital intestinal obstruction. Mg2+-ATPase activity of the myosin B was compared between the proximal dilated segment and distal segment to obstruction. Experimental colonic stenosis: In early period after surgery, proximal colons showed higher activity of myosin B ATPase than distal colons, decreasing to less than distal colon as time passed. Congenital intestinal obstruction: In three cases, whose atresia might have occurred at earlier period of gestation, proximal bowels showed less activity of myosin B ATPase than distal bowels. However, in two cases, whose atresia might have occurred at later period of gestation, and two cases with intestinal stenosis, proximal bowels indicated higher activity of myosin B ATPase than distal bowels. These data suggested that the contractibility of the proximal intestine was depending on the duration of obstruction, and it was depressed in the former patients and was accelerated in the latter patients. These results suggested that the extensive resection of dilated proximal bowel in the congenital atresia is not always necessary to obtain good postoperative intestinal dynamics at the operation of the atresial lesions which may be induced at later period of gestation. They also suggested that surgery for intestinal obstruction should be performed before the depression of intestinal contractibility to get good bowel function.

  18. Preparation and Characterization of Myosin Proteins.

    Science.gov (United States)

    Caldwell, Elizabeth; Eftink, Maurice R.

    1985-01-01

    Students complete five experimental projects at the end of a senior-level biochemistry course which involves the isolation and characterization of myosin and its water-soluble subfragments. Procedures used and results obtained are provided for such projects as viscosity and ATPase measurements and gel electrophoresis experiments. (JN)

  19. The Role of a Novel Myosin Isoform in Prostate Cancer Metastasis

    Science.gov (United States)

    2013-10-01

    2013 Accepted 14 February 2013 Available online 21 February 2013 Keywords: Myosin IC Isoforms Nucleolar localization signal Nucleolus Nucleus RNA...polymerase I Fibrillarinnt matter & 2013 Elsevier 1016/j.yexcr.2013.02.008 S, nucleolar localization ; No, nucleolus ; N, nucle bovine serum albumin; S...the nucleus, and the nucleolus . In the cytoplasm, myosin IC associ- ates with membranes and is involved in vesicle transport of membrane proteins [2

  20. Response of slow and fast muscle to hypothyroidism: maximal shortening velocity and myosin isoforms

    Science.gov (United States)

    Caiozzo, V. J.; Herrick, R. E.; Baldwin, K. M.

    1992-01-01

    This study examined both the shortening velocity and myosin isoform distribution of slow- (soleus) and fast-twitch (plantaris) skeletal muscles under hypothyroid conditions. Adult female Sprague-Dawley rats were randomly assigned to one of two groups: control (n = 7) or hypothyroid (n = 7). In both muscles, the relative contents of native slow myosin (SM) and type I myosin heavy chain (MHC) increased in response to the hypothyroid treatment. The effects were such that the hypothyroid soleus muscle expressed only the native SM and type I MHC isoforms while repressing native intermediate myosin and type IIA MHC. In the plantaris, the relative content of native SM and type I MHC isoforms increased from 5 to 13% and from 4 to 10% of the total myosin pool, respectively. Maximal shortening velocity of the soleus and plantaris as measured by the slack test decreased by 32 and 19%, respectively, in response to hypothyroidism. In contrast, maximal shortening velocity as estimated by force-velocity data decreased only in the soleus (-19%). No significant change was observed for the plantaris.

  1. C5a binding to human polymorphonuclear leukocyte plasma membrane (PMNLM) receptors

    International Nuclear Information System (INIS)

    Conway, R.G.; Mollison, K.W.; Carter, G.W.; Lane, B.

    1986-01-01

    Previous investigations of the C5a receptor have been performed using intact human PMNL. To circumvent some of the potential problems with such whole cell assays (e.g. internalization or metabolism of radioligand) the authors have developed a PMNLM binding assay. Human PMNLM were prepared by nitrogen cavitation and Percoll gradient centrifugation. Specific binding of [ 125 I]C5a to PMNLM was: high affinity, K/sub D/ = 0.6 nM; saturable, B/sub max/ = 8.7 pmol/mg protein; and reversible. Kinetic measurements agree with the K/sub D/ value obtained by Scatchard analysis. Furthermore, the binding activity of C5a correlates with biological activity as measured by myeloperoxidase release from human PMNL. Human serum C5a and recombinant C5a bind with similar affinities when measured by competition or direct binding and label the same number of sites in human PMNLM. The nonhydrolyzable GTP analog, GppNHp, induces a low affinity state of the C5a receptor (4-6 fold shift in K/sub D/) with little effect on B/sub max/. In summary, the criteria have been satisfied for identification of a biologically relevant C5a binding site in human PMNLM. Regulation of the C5a receptor and its membrane transduction mechanism(s) appears to involve guanyl nucleotides, as has been found for other chemoattractant receptors

  2. Metal cation controls phosphate release in the myosin ATPase.

    Science.gov (United States)

    Ge, Jinghua; Huang, Furong; Nesmelov, Yuri E

    2017-11-01

    Myosin is an enzyme that utilizes ATP to produce a conformational change generating a force. The kinetics of the myosin reverse recovery stroke depends on the metal cation complexed with ATP. The reverse recovery stroke is slow for MgATP and fast for MnATP. The metal ion coordinates the γ phosphate of ATP in the myosin active site. It is accepted that the reverse recovery stroke is correlated with the phosphate release; therefore, magnesium "holds" phosphate tighter than manganese. Magnesium and manganese are similar ions in terms of their chemical properties and the shell complexation; hence, we propose to use these ions to study the mechanism of the phosphate release. Analysis of octahedral complexes of magnesium and manganese show that the partial charge of magnesium is higher than that of manganese and the slightly larger size of manganese ion makes its ionic potential smaller. We hypothesize that electrostatics play a role in keeping and releasing the abstracted γ phosphate in the active site, and the stronger electric charge of magnesium ion holds γ phosphate tighter. We used stable myosin-nucleotide analog complex and Raman spectroscopy to examine the effect of the metal cation on the relative position of γ phosphate analog in the active site. We found that in the manganese complex, the γ phosphate analog is 0.01 nm further away from ADP than in the magnesium complex. We conclude that the ionic potential of the metal cation plays a role in the retention of the abstracted phosphate. © 2017 The Protein Society.

  3. Myosin heavy chain composition of single fibres from m. biceps brachii of male body builders

    DEFF Research Database (Denmark)

    Klitgaard, H; Zhou, M.-Y.; Richter, Erik

    1990-01-01

    The myosin heavy chain (MHC) composition of single fibres from m. biceps brachii of young sedentary men (28 +/- 0.4 years, mean +/- SE, n = 4) and male body builders (25 +/- 2.0 years, n = 4) was analysed with a sensitive one-dimensional electrophoretic technique. Compared with sedentary men...... expression of MHC isoforms within histochemical type II fibres of human skeletal muscle with body building. Furthermore, in human skeletal muscle differences in expression of MHC isoforms may not always be reflected in the traditional histochemical classification of types I, IIa, IIb and IIc fibres....

  4. Stretch activates myosin light chain kinase in arterial smooth muscle

    International Nuclear Information System (INIS)

    Barany, K.; Rokolya, A.; Barany, M.

    1990-01-01

    Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively

  5. Differential binding of 125I-IGF-I preparations to human fibroblast monolayers

    International Nuclear Information System (INIS)

    Conover, C.A.; Misra, P.; Hintz, R.L.; Rosenfeld, R.G.

    1988-01-01

    Specific, high affinity binding of 125 I-IGF-I to the type IIGF receptor on human fibroblast monolyaers was not altered by varying feeding schedules, serum lots, washing procedures, or incubation times and temperatures. However, markedly different competitive binding curves were obtained when different iodinated IGF-I preparations were used. Five of six radioligands bound preferentially to the type IIGF receptor on human fibroblast monolayers, with 50% displacement at 4-8 μg/l unlabelled IGF-I; with one radioligand a paradoxical 20-200% increase in 125 I-IGF-I binding was observed at low concentrations of unlabelled IGF-I, while concentrations as high as 100 μg/l IGF-I failed to displace this radioligand. The latter binding pattern cannot be accounted for by 125 -I-IGF-I binding to the type II IGF receptor. These data indicate that various radioligands may have preferential affinities for different IGF-I binding sites on human fibroblast monolayers. (author)

  6. Genes influenced by the non-muscle isoform of Myosin light chain kinase impact human cancer prognosis.

    Directory of Open Access Journals (Sweden)

    Tong Zhou

    Full Text Available The multifunctional non-muscle isoform of myosin light chain kinase (nmMLCK is critical to the rapid dynamic coordination of the cytoskeleton involved in cancer cell proliferation and migration. We identified 45 nmMLCK-influenced genes by bioinformatic filtering of genome-wide expression in wild type and nmMLCK knockout (KO mice exposed to preclinical models of murine acute inflammatory lung injury, pathologies that are well established to include nmMLCK as an essential participant. To determine whether these nmMLCK-influenced genes were relevant to human cancers, the 45 mouse genes were matched to 38 distinct human orthologs (M38 signature (GeneCards definition and underwent Kaplan-Meier survival analysis in training and validation cohorts. These studies revealed that in training cohorts, the M38 signature successfully identified cancer patients with poor overall survival in breast cancer (P<0.001, colon cancer (P<0.001, glioma (P<0.001, and lung cancer (P<0.001. In validation cohorts, the M38 signature demonstrated significantly reduced overall survival for high-score patients of breast cancer (P = 0.002, colon cancer (P = 0.035, glioma (P = 0.023, and lung cancer (P = 0.023. The association between M38 risk score and overall survival was confirmed by univariate Cox proportional hazard analysis of overall survival in the both training and validation cohorts. This study, providing a novel prognostic cancer gene signature derived from a murine model of nmMLCK-associated lung inflammation, strongly supports nmMLCK-involved pathways in tumor growth and progression in human cancers and nmMLCK as an attractive candidate molecular target in both inflammatory and neoplastic processes.

  7. Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Fulcher, F. Kent; Smith, Bethany T.; Russ, Misty; Patel, Yashomati M.

    2008-01-01

    Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity

  8. Myosin X is recruited to nascent focal adhesions at the leading edge and induces multi-cycle filopodial elongation.

    Science.gov (United States)

    He, Kangmin; Sakai, Tsuyoshi; Tsukasaki, Yoshikazu; Watanabe, Tomonobu M; Ikebe, Mitsuo

    2017-10-20

    Filopodia protrude from the leading edge of cells and play important roles in cell motility. Here we report the mechanism of myosin X (encoded by Myo10)-induced multi-cycle filopodia extension. We found that actin, Arp2/3, vinculin and integrin-β first accumulated at the cell's leading edge. Myosin X was then gathered at these sites, gradually clustered by lateral movement, and subsequently initiated filopodia formation. During filopodia extension, we found the translocation of Arp2/3 and integrin-β along filopodia. Arp2/3 and integrin-β then became localized at the tip of filopodia, from where myosin X initiated the second extension of filopodia with a change in extension direction, thus producing long filopodia. Elimination of integrin-β, Arp2/3 and vinculin by siRNA significantly attenuated the myosin-X-induced long filopodia formation. We propose the following mechanism. Myosin X accumulates at nascent focal adhesions at the cell's leading edge, where myosin X promotes actin convergence to create the base of filopodia. Then myosin X moves to the filopodia tip and attracts integrin-β and Arp2/3 for further actin nucleation. The tip-located myosin X then initiates the second cycle of filopodia elongation to produce the long filopodia.

  9. Expression of smooth muscle and non-muscle myosin heavy chain isoforms in cultured vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Rovner, A.S.; Murphy, R.A.; Owens, G.K.

    1986-01-01

    Immunocytochemical studies of cultured smooth muscle cells (SMCs) have disagreed on the nature of myosin expression. This investigation was undertaken to test for the presence of heterogeneous myosin heavy chain (MHC) isoforms in cell culture as a possible explanation for these results. Previously, Rovner et al. detected two MHCs in intact smooth muscles which differed in molecular weight by ca. 4000 daltons (SM1 and SM2) using a 3-4% acrylamide gradient SDS gel system. When sub-confluent primary cultures of rat aorta SMCs were assayed by this system, SM1 and SM2 were seen, along with large amounts of a third, unique MHC, NM, which closely resembled the MHC from human platelet in size and antigenicity. Data from 35 S-methionine autoradiograms showed that the log growth phase SMC cultures were producing almost exclusively NM, but the growth arrest, post-confluent cultures synthesized increased relative amounts of the SM MHC forms and contained comparable amounts of SM1, SM2, and NM. The same patterns of MHC synthesis were seen in sub-passaged SMCs. The expression of the SM-specific forms of myosin in quiescent, post-confluent cultures parallels that of smooth muscle actin suggesting that density induced growth arrest promotes cytodifferentiation in cultured vascular SMCs

  10. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    International Nuclear Information System (INIS)

    Maruta, H.; Korn, E.D.

    1981-01-01

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0 0 C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain

  11. Myosins and DYNLL1/LC8 in the honey bee (Apis mellifera L.) brain.

    Science.gov (United States)

    Calábria, Luciana Karen; Peixoto, Pablo Marco Veras; Passos Lima, Andreia Barcelos; Peixoto, Leonardo Gomes; de Moraes, Viviane Rodrigues Alves; Teixeira, Renata Roland; Dos Santos, Claudia Tavares; E Silva, Letícia Oliveira; da Silva, Maria de Fátima Rodrigues; dos Santos, Ana Alice Diniz; Garcia-Cairasco, Norberto; Martins, Antônio Roberto; Espreafico, Enilza Maria; Espindola, Foued Salmen

    2011-09-01

    Honey bees have brain structures with specialized and developed systems of communication that account for memory, learning capacity and behavioral organization with a set of genes homologous to vertebrate genes. Many microtubule- and actin-based molecular motors are involved in axonal/dendritic transport. Myosin-Va is present in the honey bee Apis mellifera nervous system of the larvae and adult castes and subcastes. DYNLL1/LC8 and myosin-IIb, -VI and -IXb have also been detected in the adult brain. SNARE proteins, such as CaMKII, clathrin, syntaxin, SNAP25, munc18, synaptophysin and synaptotagmin, are also expressed in the honey bee brain. Honey bee myosin-Va displayed ATP-dependent solubility and was associated with DYNLL1/LC8 and SNARE proteins in the membrane vesicle-enriched fraction. Myosin-Va expression was also decreased after the intracerebral injection of melittin and NMDA. The immunolocalization of myosin-Va and -IV, DYNLL1/LC8, and synaptophysin in mushroom bodies, and optical and antennal lobes was compared with the brain morphology based on Neo-Timm histochemistry and revealed a distinct and punctate distribution. This result suggested that the pattern of localization is associated with neuron function. Therefore, our data indicated that the roles of myosins, DYNLL1/LC8, and SNARE proteins in the nervous and visual systems of honey bees should be further studied under different developmental, caste and behavioral conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. The fungal myosin I is essential for Fusarium toxisome formation.

    Directory of Open Access Journals (Sweden)

    Guangfei Tang

    2018-01-01

    Full Text Available Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1, the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi.

  13. The fungal myosin I is essential for Fusarium toxisome formation.

    Science.gov (United States)

    Tang, Guangfei; Chen, Yun; Xu, Jin-Rong; Kistler, H Corby; Ma, Zhonghua

    2018-01-01

    Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1), the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON) biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi.

  14. Phosphorylated peptides occur in a non-helical portion of the tail of a catch muscle myosin

    International Nuclear Information System (INIS)

    Castellani, L.; Elliott, B.W. Jr.; Cohen, C.

    1987-01-01

    Myosin from a molluscan catch muscle (the Anterior Byssus Retractor (ABRM) of Mytilus edulis) is unusual in being phosphorylated in the rod by an endogenous heavy-chain kinase. This phosphorylation enhances myosin solubility at low ionic strength and induces molecular folding of the myosin tail. Papain and chymotryptic cleavage of this myosin, phosphorylated with [γ- 32 P]ATP, indicates that the phosphorylated residues are associated with the carboxy-terminal end of the light meromyosin. Ion-exchange and reverse-phase HPLC of radiolabeled chymotryptic peptides allow the isolation of two different peptides with high specific activity. One of these peptides is rich in lysine and arginine residues, a finding consistent with the observation that basic residues often determine the substrate specificity of protein kinases. The second peptide contains proline residues. Taken together, these results suggest that, as in the case of Acanthamoeba myosin, phosphorylation occurs in a nonhelical portion of the rod that may also control solubility. Identification of the residues that are phosphorylated and their location in the rod may reveal how the phosphorylation-dependent changes observed in the myosin in vitro are related to changes in intermolecular interactions in the thick filaments in vivo

  15. The Effect of Experimental Hyperthyroidism on Characteristics of Actin-Myosin Interaction in Fast and Slow Skeletal Muscles.

    Science.gov (United States)

    Kopylova, G V; Shchepkin, D V; Bershitsky, S Y

    2018-05-01

    The molecular mechanism of the failure of contractile function of skeletal muscles caused by oxidative damage to myosin in hyperthyroidism is not fully understood. Using an in vitro motility assay, we studied the effect of myosin damage caused by oxidative stress in experimental hyperthyroidism on the actin-myosin interaction and its regulation by calcium. We found that hyperthyroidism-induced oxidation of myosin is accompanied by a decrease in the sliding velocity of the regulated thin filaments in the in vitro motility assay, and this effect is increased with the duration of the pathological process.

  16. Binding of tissue plasminogen activator to human umbilical vein endothelial cells

    International Nuclear Information System (INIS)

    Beebe, D.P.

    1987-01-01

    The binding of purified, recombinant tissue plasminogen activator (tPA) to human umbilical vein endothelial cells (HUVEC) was studied in vitro using immunofluorescence as well as radiolabeled tPA. Immunofluorescence was performed on HUVEC grown on round glass coverslips using rabbit anti-human tPA and fluorescein-conjugated anti-rabbit immunoglobulin. Positive fluorescence was observed only after incubation of HUVEC with tPA. HUVEC were grown to confluence in 24-well tissue culture plates, washed, and incubated with a constant amount of 125 I-tPA and various concentrations of unlabeled tPA. The binding of tPA to HUVEC was found to be specific, saturable, and reversible. Scatchard analysis yielded as equilibrium constant (K/sub eq/) of 4.2 x 10 6 M -1 and 1.2 x 10 7 binding sites per cell. Binding was inhibited by positively charged amino acids and by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone but not by carbohydrates including mannose, galactose, N-acetyl glucosamine and N-acetyl galactosamine. Neat human plasma abrogates but does not totally inhibit binding of tPA to HUVEC. Binding was neither enhanced nor inhibited by fibronectin. Although the affinity of binding of tPA to HUVEC is low, the endothelial cell may be involved in regulating plasma levels of tPA in vivo which may have therapeutic significance

  17. Characteristics of high affinity and low affinity adenosine binding sites in human cerebral cortex

    International Nuclear Information System (INIS)

    John, D.; Fox, I.V.

    1986-01-01

    The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A 1 and A 2 adenosine binding sites. The ligands used were 2-chloro(8- 3 H) adenosine and N 6 -(adenine-2, 8- 3 H) cyclohexayladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The kinetic constant determinations from binding studies of the adenosine receptor are presented. Utilizing tritium-cyclohexyladenosine as ligand the authors observed evidence for a high affinity binding site in human brain cortical membranes with a kd of 5 nM

  18. Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics

    International Nuclear Information System (INIS)

    Ayyagari, R.R.; Khan-Dawood, F.S.

    1987-01-01

    Epidermal growth factor receptors are present in many reproductive tissues but have not been demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2 hours at 4 degrees C with 500 micrograms plasma membrane protein and 140 femtomol 125 I-epidermal growth factor per incubate. The number (mean +/- SEM) of epidermal growth factor binding sites was 12.34 +/- 2.99 X 10(-19) mol/micrograms protein; the dissociation constant was 2.26 +/- 0.56 X 10(-9) mol/L; the association constant was 0.59 +/- 0.12 X 10(9) L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum. The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but epidermal growth factor may be involved in intragonadal regulation of luteal function

  19. Human serum albumin binding of certain antimalarials

    Science.gov (United States)

    Marković, Olivera S.; Cvijetić, Ilija N.; Zlatović, Mario V.; Opsenica, Igor M.; Konstantinović, Jelena M.; Terzić Jovanović, Nataša V.; Šolaja, Bogdan A.; Verbić, Tatjana Ž.

    2018-03-01

    Interactions between eight in-house synthesized aminoquinolines, along with well-known chloroquine, and human serum albumin (HSA) have been studied by fluorescence spectroscopy. The synthesized aminoquinolines, despite being structurally diverse, were found to be very potent antimalarials. Fluorescence measurements indicate that three compounds having additional thiophene or benzothiophene substructure bind more strongly to HSA than other studied compounds. Competitive binding experiments indicate that these three compounds bind significantly stronger to warfarin compared to diazepam binding site. Fluorescence quenching at three temperatures (20, 25, and 37 °C) was analyzed using classical Stern-Volmer equation, and a static quenching mechanism was proposed. The enthalpy and entropy changes upon sulphur-containing compound-HSA interactions were calculated using Van't Hoff equation. Positive values of enthalpy and entropy changes indicate that non-specific, hydrophobic interactions are the main contributors to HSA-compound interaction. Molecular docking and calculated lipophilicity descriptors indicate the same, pointing out that the increased lipophilicity of sulphur-containing compounds might be a reason for their better binding to HSA. Obtained results might contribute to design of novel derivatives with improved pharmacokinetic properties and drug efficacy.

  20. Thermodynamic analysis of allosamidin binding to the human chitotriosidase

    Energy Technology Data Exchange (ETDEWEB)

    Eide, Kristine Bistrup; Lundmark, Silje Thoresen [Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås (Norway); Sakuda, Shohei [Department of Applied Biological Chemistry, University of Tokyo, Bunkyo-Ku, Tokyo 113 (Japan); Sørlie, Morten, E-mail: morten.sorlie@umb.no [Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås (Norway)

    2013-08-10

    Highlights: • Large differences in thermodynamic signatures for family 18 chitinase inhibition. • Allosamidin binds tight to HCHT. • Binding driven by enthalpy change and desolvation. - Abstract: Human chitotriosidase (HCHT) is one of two active family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the innate human defense mechanism against fungal parasites. Recently, it has been shown that levels of HCHT bioactivity and protein are significantly increased in the circulation and lungs of systemic sclerosis patients and for this reason is a suggested therapeutic target. For this reason, we have undertaken a detailed thermodynamic investigation using isothermal titration calorimetry of the binding interaction of HCHT with the well-known family 18 chitinase inhibitor allosamidin. The binding is shown to be strong (K{sub d} = 0.20 ± 0.03 μM and ΔG{sub r}° = −38.9 ± 0.4 kJ/mol) and driven by favorable changes in enthalpy (ΔH{sub r}° = −50.2 ± 1.2 kJ/mol) and solvation entropy (−TΔS{sub solv}° = −41.8 ± 4.4 kJ/mol). It is accompanied with a large penalty in conformational entropy change (−TΔS{sub conf}° = 43.1 ± 4.2 kJ/mol)

  1. Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

    Science.gov (United States)

    Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, Volker; Rülicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Önder; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Hozák, Pavel

    2013-01-01

    Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

  2. Characterisation of the human NMDA receptor subunit NR3A glycine binding site

    DEFF Research Database (Denmark)

    Nilsson, A; Duan, J; Mo-Boquist, L-L

    2007-01-01

    In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277...

  3. β-Arrestin regulation of myosin light chain phosphorylation promotes AT1aR-mediated cell contraction and migration.

    Directory of Open Access Journals (Sweden)

    Elie Simard

    Full Text Available Over the last decade, it has been established that G-protein-coupled receptors (GPCRs signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC, which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1 as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR, MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM. Furthermore, by employing the β-arrestin biased ligand [Sar(1,Ile(4,Ile(8]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.

  4. A novel role for myosin II in insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Steimle, Paul A.; Kent Fulcher, F.; Patel, Yashomati M.

    2005-01-01

    Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles from an intracellular pool to the plasma membrane. The studies presented here show that inhibition of myosin II activity impairs GLUT4-mediated glucose uptake but not GLUT4 translocation to the plasma membrane. We also show that adipocytes express both myosin IIA and IIB isoforms, and that myosin IIA is recruited to the plasma membrane upon insulin stimulation. Taken together, the data presented here represent the first demonstration that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. Based on our findings, we hypothesize that myosin II is activated upon insulin stimulation and recruited to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. The identification of myosin II as a key component of GLUT4-mediated glucose uptake represents an important advance in our understanding of the mechanisms regulating glucose homeostasis

  5. Myosin-II sets the optimal response time scale of chemotactic amoeba

    Science.gov (United States)

    Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Bodenschatz, Eberhard; Beta, Carsten

    2014-03-01

    The response dynamics of the actin cytoskeleton to external chemical stimuli plays a fundamental role in numerous cellular functions. One of the key players that governs the dynamics of the actin network is the motor protein myosin-II. Here we investigate the role of myosin-II in the response of the actin system to external stimuli. We used a microfluidic device in combination with a photoactivatable chemoattractant to apply stimuli to individual cells with high temporal resolution. We directly compare the actin dynamics in Dictyostelium discodelium wild type (WT) cells to a knockout mutant that is deficient in myosin-II (MNL). Similar to the WT a small population of MNL cells showed self-sustained oscillations even in absence of external stimuli. The actin response of MNL cells to a short pulse of chemoattractant resembles WT during the first 15 sec but is significantly delayed afterward. The amplitude of the dominant peak in the power spectrum from the response time series of MNL cells to periodic stimuli with varying period showed a clear resonance peak at a forcing period of 36 sec, which is significantly delayed as compared to the resonance at 20 sec found for the WT. This shift indicates an important role of myosin-II in setting the response time scale of motile amoeba. Institute of Physics und Astronomy, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, Germany.

  6. Engineering Circular Gliding of Actin Filaments Along Myosin-Patterned DNA Nanotube Rings To Study Long-Term Actin-Myosin Behaviors.

    Science.gov (United States)

    Hariadi, Rizal F; Appukutty, Abhinav J; Sivaramakrishnan, Sivaraj

    2016-09-27

    Nature has evolved molecular motors that are critical in cellular processes occurring over broad time scales, ranging from seconds to years. Despite the importance of the long-term behavior of molecular machines, topics such as enzymatic lifetime are underexplored due to the lack of a suitable approach for monitoring motor activity over long time periods. Here, we developed an "O"-shaped Myosin Empowered Gliding Assay (OMEGA) that utilizes engineered micron-scale DNA nanotube rings with precise arrangements of myosin VI to trap gliding actin filaments. This circular gliding assay platform allows the same individual actin filament to glide over the same myosin ensemble (50-1000 motors per ring) multiple times. First, we systematically characterized the formation of DNA nanotubes rings with 4, 6, 8, and 10 helix circumferences. Individual actin filaments glide along the nanotube rings with high processivity for up to 12.8 revolutions or 11 min in run time. We then show actin gliding speed is robust to variation in motor number and independent of ring curvature within our sample space (ring diameter of 0.5-4 μm). As a model application of OMEGA, we then analyze motor-based mechanical influence on "stop-and-go" gliding behavior of actin filaments, revealing that the stop-to-go transition probability is dependent on motor flexibility. Our circular gliding assay may provide a closed-loop platform for monitoring long-term behavior of broad classes of molecular motors and enable characterization of motor robustness and long time scale nanomechanical processes.

  7. The Duffy binding protein (PkDBPαII) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

    Science.gov (United States)

    Lim, Khai Lone; Amir, Amirah; Lau, Yee Ling; Fong, Mun Yik

    2017-08-11

    The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBPαII haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaque (Macaca fascicularis) erythrocytes was investigated. The PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes. Anti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly, the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBPαII haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P < 0.0001). However, no difference in binding activity level was seen in the binding assay using M. fascicularis erythrocytes. This study is the first report of phenotypic difference between PkDBPαII haplotypes. The biological implication of this finding is yet to be determined. Therefore, further studies need to be carried out to determine whether this differential binding level can be associated with severity of knowlesi malaria in human.

  8. Gonadotropin binding sites in human ovarian follicles and corpora lutea during the menstrual cycle

    Energy Technology Data Exchange (ETDEWEB)

    Shima, K.; Kitayama, S.; Nakano, R.

    1987-05-01

    Gonadotropin binding sites were localized by autoradiography after incubation of human ovarian sections with /sup 125/I-labeled gonadotropins. The binding sites for /sup 125/I-labeled human follicle-stimulating hormone (/sup 125/I-hFSH) were identified in the granulosa cells and in the newly formed corpora lutea. The /sup 125/I-labeled human luteinizing hormone (/sup 125/I-hLH) binding to the thecal cells increased during follicular maturation, and a dramatic increase was preferentially observed in the granulosa cells of the large preovulatory follicle. In the corpora lutea, the binding of /sup 125/I-hLH increased from the early luteal phase and decreased toward the late luteal phase. The changes in 3 beta-hydroxysteroid dehydrogenase activity in the corpora lutea corresponded to the /sup 125/I-hLH binding. Thus, the changes in gonadotropin binding sites in the follicles and corpora lutea during the menstrual cycle may help in some important way to regulate human ovarian function.

  9. Radioimmunoassay of myosin heavy beta chains in human serum for the evaluation of the size of myocardial infarction: correlation with myocardial Tl-201 SPECT and cardiac angioscintigraphy

    International Nuclear Information System (INIS)

    Facello, A.; Gries, P.; Demangeat, C.; Brunot, B.; Roul, G.; Demangeat, J.L.; Moulichon, M.; Bareiss, P.; Sacrez, A.; Constantinesco, A.

    1990-01-01

    To determine the relationship between serum levels of myosin heavy beta chains assessed by an IRMA technique and other radionuclide and enzymatic parameters in the evaluation of the size of myocardial infarction, we studied 22 patients with acute myocardial infarction. Blood samples taken daily between 1st to 13th day of evolution allow the determination of peak and integral of myosine release that showed a good correlation (p [fr

  10. Binding of radioiodinated human. beta. -endorphin to serum proteins from rats and humans, determined by several methods

    Energy Technology Data Exchange (ETDEWEB)

    Sato, H.; Sugiyama, Y.; Sawada, Y.; Iga, T.; Hanano, M.

    1985-10-07

    Binding of immunoreactive radioiodinated human ..beta..-endorphin (/sup 125/I-..beta..-EP) to rat serum was demonstrated by gel filtration of /sup 125/I-..beta..-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, ..cap alpha../sub 2/- and ..beta../sub 2/-macroglobulins, and the second peak at the fraction of albumin. Binding of /sup 125/I-..beta..-EP to albumin was directly proved by gel filtration of /sup 125/I-..beta..-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of /sup 125/I-..beta..-EP with serum proteins, because of the intense nonspecific adsorption to the semi-permeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of /sup 125/I-..beta..-EP in sera from rats and humans, the authors utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of /sup 125/I-..beta..-EP in rat serum. Binding of /sup 125/I-..beta..-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of /sup 125/I-..beta..-EP was concentration independent over the concentration range studied (1-1000 nM). 23 references, 4 figures, 1 table.

  11. Thermodynamic parameters for binding of fatty acids to human serum albumin

    DEFF Research Database (Denmark)

    Pedersen, A O; Honoré, B; Brodersen, R

    1990-01-01

    Binding of laurate and myristate anions to human serum albumin has been studied over a range of temperatures, 5-37 degrees C, at pH 7.4. The binding curves indicate that the strength of binding of the first few molecules of fatty acid to albumin (r less than 5) decreases with increasing temperatu...

  12. Expression, purification, crystallization and structure of human adipocyte lipid-binding protein (aP2)

    International Nuclear Information System (INIS)

    Marr, Eric; Tardie, Mark; Carty, Maynard; Brown Phillips, Tracy; Wang, Ing-Kae; Soeller, Walt; Qiu, Xiayang; Karam, George

    2006-01-01

    The crystal structure of human adipocyte lipid-binding protein (aP2) with a bound palmitate is reported at 1.5 Å resolution. Human adipocyte lipid-binding protein (aP2) belongs to a family of intracellular lipid-binding proteins involved in the transport and storage of lipids. Here, the crystal structure of human aP2 with a bound palmitate is described at 1.5 Å resolution. Unlike the known crystal structure of murine aP2 in complex with palmitate, this structure shows that the fatty acid is in a folded conformation and that the loop containing Phe57 acts as a lid to regulate ligand binding by excluding solvent exposure to the central binding cavity

  13. Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes

    OpenAIRE

    Bon?ina, Matja?; Podlipnik, ?rtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

    2015-01-01

    Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolini...

  14. Identification and characterization of riboflavin-binding proteins in human circulation

    International Nuclear Information System (INIS)

    Innis-Whitehouse, W.S.A.

    1988-01-01

    Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis and binding was observed to vary over a greater than 10-fold range. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly, although FMN and photo-chemical degradation products were more tightly bound. Most of the binding occurred in the gamma-globulin fraction and was attributed to immunoglobulins because the binding proteins and immunoglobulins copurified using various methods, were removed by treatment of plasma with protein A-agarose, and were coincident upon immuno-electrophoresis followed by autoradiography to detect [2- 14 C]-riboflavin. Binding differences among plasma samples were reflected in the binding recovered with the immunoglobulin fractions; however, there was not a direct relationship between the amount of immunoglobulin and the amount of [2- 14 C]riboflavin bound. Hence, it appeared that the binding was due to a subfraction of immunoglobulins

  15. Studies on binding of radiolabeled thyrotropin to cultured human thyroid cells

    International Nuclear Information System (INIS)

    Yamamoto, M.; Rapoport, B.

    1978-01-01

    A line of cultured human thyroid adenoma cells was used in a study designed to compare the stimulatory effect of TSH on cellular cAMP generation with the binding of radiolabeled TSH to the cells. At 37 C, specific binding of [ 125 I]TSH to suspensions of thyroid cells was maximal at 20 min and was reversed by the addition of excess TSH. Unlike the generation of cellular cAMP in response to TSH stimulation, which was maximal at pH 7.5, the binding of [ 125 ]TSH to the cells was maximal at pH 5.5 and progressively declined up to pH 8.5. Increasing NaCl concentrations progressively inhibited cellular binding of TSH; at physiological salt concentrations, almost no TSH binding was detectable. Competitive inhibition studies of [ 125 I]TSH binding to cells revealed a binding site with a dissociation constant of 5.5 x 10 -8 M at pH 7.4. GH, PRL, hCG, FSH, insulin, and glucagon did not compete with [ 125 I)TSH binding. ACTH, however, was a potent inhibitor of [ 125 I]TSH binding. Despite this inhibitory effect on TSH binding, ACTH had little or no effect on cellular cAMP generation. High concentrations of ACTH did not inhibit the biological effect of TSH on cAMP generation. Specific binding of [ 125 I]TSH to empty plastic culture dishes was time dependent, reversible, and displayed a hormonal specificity identical to binding to thyroid cells. The effects of pH and NaCl concentrations on TSH binding to dishes were similarbut not identical to those on cellular binding. This study raises serious questions as to the biological significance of [ 125 I]TSH binding to cultured human thyroid cells

  16. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    Science.gov (United States)

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  17. Effect of ageing on the myosin heavy chain composition of the human sternocleidomastoid muscle.

    Science.gov (United States)

    Meznaric, M; Eržen, I; Karen, P; Cvetko, E

    2018-03-01

    The myosin heavy chain (MyHC) composition of ageing limb muscles is transformed into a slower phenotype and expresses fast-twitch fibre type atrophy, presumably due to age-related motor unit remodelling and a change in the patterns of physical activity. It is not known if ageing affects the sternocleidomastoid muscle (SCM) in a similar way. The goal of the study was to analyze the MyHC composition and the size of muscle fibres in the ageing SCM by immunohistochemical methods and quantitative analysis and stereology using our own software for morphometry. We hypothesize that with ageing the MyHC composition of SCM transforms similarly as in ageing limb muscles, but the size of the muscle fibres is less effected as in limb muscles. The study was performed on the autopsy samples of the SCM in 12 older males. The results were compared with those published in our previous study on 15 young adult males. An ageing SCM transforms into a slower MyHC profile: the percentage of slow-twitch fibres is enhanced (numerical proportion 44.6 vs. 31.5%, Pfibres is diminished (numerical proportion 14.1 vs. 26.8%, Pfast-twitch fibres expressing MyHC-2a and 2x is smaller (50.6 vs. 63.5%, Pfibres expressing the fastest myosin isoform MyHC-2x is smaller too (19.0 vs. 34.5%, Pfibres expressing the fastest MyHC-2x provide circumstantial evidence for: (i) more fast-twitch than slow-twitch motor units being lost; and (ii) reinnervation by the surviving motor units. There appears to be no significant influence on muscle fibre size, which is congruent with relatively unchanged SCM activity during life. Copyright © 2017 Elsevier GmbH. All rights reserved.

  18. New approaches to high-throughput structure characterization of SH3 complexes: the example of Myosin-3 and Myosin-5 SH3 domains from S. cerevisiae.

    Science.gov (United States)

    Musi, Valeria; Birdsall, Berry; Fernandez-Ballester, Gregorio; Guerrini, Remo; Salvatori, Severo; Serrano, Luis; Pastore, Annalisa

    2006-04-01

    SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.

  19. Multiple binding of bilirubin to human serum albumin and cobinding with laurate

    DEFF Research Database (Denmark)

    Sato, H; Honoré, B; Brodersen, R

    1988-01-01

    Numerical analysis of multiple binding of two ligands to one carrier has been accomplished, using the principle of several sets of acceptable binding constants, with bilirubin-laurate-albumin as an example. Binding of bilirubin to defatted human serum albumin was investigated by a spectroscopic...

  20. Palmitate and stearate binding to human serum albumin. Determination of relative binding constants

    DEFF Research Database (Denmark)

    Vorum, H; Fisker, K; Honoré, B

    1997-01-01

    Multiple binding equilibria of two apparently insoluble ligands, palmitate and stearate, to defatted human serum albumin were studied in a 66 mM sodium phosphate buffer (pH 7.4) at 37 degrees C, by determination of dialytic exchange rates of ligands among identical equilibrium solutions. The expe...

  1. Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover, contraction, and stress-stabilization.

    Science.gov (United States)

    Nie, Wei; Wei, Ming-Tzo; Ou-Yang, H Daniel; Jedlicka, Sabrina S; Vavylonis, Dimitrios

    2015-01-01

    The morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked fibers along the contacting surface. The motor activity and minifilament assembly of non-muscle myosin-II is an important component of cortical cytoskeletal remodeling during mechanosensing. We used experiments and computational modeling to study cortical myosin-II dynamics in adhered cells. Confocal microscopy was used to image the medial cell cortex of HeLa cells stably expressing myosin regulatory light chain tagged with GFP (MRLC-GFP). The distribution of MRLC-GFP fibers and focal adhesions was classified into three types of network morphologies. Time-lapse movies show: myosin foci appearance and disappearance; aligning and contraction; stabilization upon alignment. Addition of blebbistatin, which perturbs myosin motor activity, leads to a reorganization of the cortical networks and to a reduction of contractile motions. We quantified the kinetics of contraction, disassembly and reassembly of myosin networks using spatio-temporal image correlation spectroscopy (STICS). Coarse-grained numerical simulations include bipolar minifilaments that contract and align through specified interactions as basic elements. After assuming that minifilament turnover decreases with increasing contractile stress, the simulations reproduce stress-dependent fiber formation in between focal adhesions above a threshold myosin concentration. The STICS correlation function in simulations matches the function measured in experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. © 2015 Wiley Periodicals, Inc.

  2. Multiple binding modes of ibuprofen in human serum albumin identified by absolute binding free energy calculations

    KAUST Repository

    Evoli, Stefania

    2016-11-10

    Human serum albumin possesses multiple binding sites and transports a wide range of ligands that include the anti-inflammatory drug ibuprofen. A complete map of the binding sites of ibuprofen in albumin is difficult to obtain in traditional experiments, because of the structural adaptability of this protein in accommodating small ligands. In this work, we provide a set of predictions covering the geometry, affinity of binding and protonation state for the pharmaceutically most active form (S-isomer) of ibuprofen to albumin, by using absolute binding free energy calculations in combination with classical molecular dynamics (MD) simulations and molecular docking. The most favorable binding modes correctly reproduce several experimentally identified binding locations, which include the two Sudlow\\'s drug sites (DS2 and DS1) and the fatty acid binding sites 6 and 2 (FA6 and FA2). Previously unknown details of the binding conformations were revealed for some of them, and formerly undetected binding modes were found in other protein sites. The calculated binding affinities exhibit trends which seem to agree with the available experimental data, and drastically degrade when the ligand is modeled in a protonated (neutral) state, indicating that ibuprofen associates with albumin preferentially in its charged form. These findings provide a detailed description of the binding of ibuprofen, help to explain a wide range of results reported in the literature in the last decades, and demonstrate the possibility of using simulation methods to predict ligand binding to albumin.

  3. Stress generation by myosin minifilaments in actin bundles

    International Nuclear Information System (INIS)

    Dasanayake, Nilushi L; Carlsson, Anders E

    2013-01-01

    Forces and stresses generated by the action of myosin minifilaments are analyzed in idealized computer-generated actin bundles, and compared to results for isotropic actin networks. The bundles are generated as random collections of actin filaments in two dimensions with constrained orientations, crosslinked and attached to two fixed walls. Myosin minifilaments are placed on actin filament pairs and allowed to move and deform the network so that it exerts forces on the walls. The vast majority of simulation runs end with contractile minifilament stress, because minifilaments rotate into energetically stable contractile configurations. This process is aided by the bending and stretching of actin filaments, which accomodate minifilament rotation. Stresses for bundles are greater than those for isotropic networks, and antiparallel filaments generate more tension than parallel filaments. The forces transmitted by the actin network to the walls of the simulation cell often exceed the tension in the minifilament itself. (paper)

  4. Human myometrial adrenergic receptors: identification of the beta-adrenergic receptor by [3H]dihydroalprenolol binding

    International Nuclear Information System (INIS)

    Hayashida, D.N.; Leung, R.; Goldfien, A.; Roberts, J.M.

    1982-01-01

    The radioactive beta-adrenergic antagonist [ 3 H] dihydroalprenolol (DHA) binds to particulate preparations of human myometrium in a manner compatible with binding to the beta-adrenergic receptor. The binding of DHA is rapid (attaining equilibrium in 12 minutes), readily reversible (half time = 16 minutes), high affinity (K/sub D/ = 0.50 nM), low capacity (Bmax = 70 fmoles/mg of protein), and stereoselective ([-]-propranolol is 100 times as potent as [+] -propranolol in inhibiting DHA binding). Adrenergic agonists competed for DHA binding sites in a manner compatible with beta-adrenergic interactions and mirrored β 2 pharmacologic potencies: isoproterenol > epinephrine >> norepinephrine. Studies in which zinterol, a β 2 -adrenergic agonist, competed for DHA binding sites in human myometrial particulate indicated that at least 87% of the beta-adrenergic receptors present are β 2 -adrenergic receptors. Binding of DHA to human myometrial beta-adrenergic receptors provides a tool which may be used in the examination of gonadal hormonal modification of adrenergic response in human uterus as well as in the analysis of beta-adrenergic agents as potentially useful tocolytic agents

  5. Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors

    International Nuclear Information System (INIS)

    Mueller, H.; Fehr, J.

    1986-01-01

    The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexes or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125 I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PNM) with a K/sub D/ of 10.5 +/- 5.7 x 10 -8 M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 x 10 6 . Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic simulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposits at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus

  6. Genome-wide binding and transcriptome analysis of human farnesoid X receptor in primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Le Zhan

    Full Text Available Farnesoid X receptor (FXR, NR1H4 is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing (ChIP-seq, with results indicating that FXR may be involved in regulating diverse pathways in liver. However, limited information exists for the functions of human FXR and the suitability of using murine models to study human FXR functions.In the current study, we performed ChIP-seq in primary human hepatocytes (PHHs treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq and RNA microarray were performed for GW4064 or control treated PHHs and wild type mouse livers, respectively.ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment.In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions.

  7. Mechanical Defects of Muscle Fibers with Myosin Light Chain Mutants that Cause Cardiomyopathy

    OpenAIRE

    Roopnarine, Osha

    2003-01-01

    Familial hypertrophic cardiomyopathy is a disease caused by single mutations in several sarcomeric proteins, including the human myosin ventricular regulatory light chain (vRLC). The effects of four of these mutations (A13T, F18L, E22K, and P95A) in vRLC on force generation were determined as a function of Ca2+ concentration. The endogenous RLC was removed from skinned rabbit psoas muscle fibers, and replaced with either rat wildtype vRLC or recombinant rat vRLC (G13T, F18L, E22K, and P95A). ...

  8. Fluorescent modification and orientation of myosin sulfhydryl 2 in skeletal muscle fibers

    International Nuclear Information System (INIS)

    Ajtai, K.; Burghardt, T.P.

    1989-01-01

    The authors describe a protocol for the selective covalent labeling of the sulfhydryl 2 (SH2) on the myosin cross-bridge in glycerinated muscle fibers using the sulfhydryl-selective label 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The protocol promotes the specificity of IANBD by using the ability to protect sulfhydryl 1 (SH1) from modification by binding the cross-bridge to the actin filament and using cross-bridge-bound MgADP to promote the accessibility of SH2. They determined the specificity of the probe using fluorescence gel scanning of fiber-extracted proteins to isolate the probe on myosin subfragment 1 (S1), limited proteolysis of the purified S1 to isolate the probe on the 20-kilodalton fragment of S1, and titration of the free SH1's on purified S1 using the radiolabeled SH1-specific reagent [ 14 C]iodoacetamide or enzymatic activity measurements. They characterized the angular distribution of the IANBD on cross-bridges in fibers when the fibers are in rigor, in relaxation, in the presence of MgADP, and in isometric contraction using wavelength-dependent fluorescence polarization. They find that the SH2 probe distinguishes the different states of the fiber such that rigor and MgADP are ordered and maintain a similar orientation throughout the excitation wavelength domain. The relaxed cross-bridge is ordered and has an orientation that is distinct from the orientation of the cross-bridge in rigor and MgADP over the entire wavelength domain. The active isometric cross-bridge is also oriented differently from the other states, suggesting the presence of a predominant actin-bound cross-bridge state that precedes the power stroke during muscle contraction

  9. Preliminary structural characterization of human SOUL, a haem-binding protein

    International Nuclear Information System (INIS)

    Freire, Filipe; Romão, Maria João; Macedo, Anjos L.; Aveiro, Susana S.; Goodfellow, Brian J.; Carvalho, Ana Luísa

    2009-01-01

    This manuscript describes the overexpression, purification and crystallization of human SOUL protein (hSOUL). hSOUL is a 23 kDa haem-binding protein that was first identified as the PP23 protein isolated from human full-term placenta. Human SOUL (hSOUL) is a 23 kDa haem-binding protein that was first identified as the PP 23 protein isolated from human full-term placentas. Here, the overexpression, purification and crystallization of hSOUL are reported. The crystals belonged to space group P6 4 22, with unit-cell parameters a = b = 145, c = 60 Å and one protein molecule in the asymmetric unit. X-ray diffraction data were collected to 3.5 Å resolution at the ESRF. A preliminary model of the three-dimensional structure of hSOUL was obtained by molecular replacement using the structures of murine p22HBP, obtained by solution NMR, as search models

  10. Antioxidant flavonoids bind human serum albumin

    Science.gov (United States)

    Kanakis, C. D.; Tarantilis, P. A.; Polissiou, M. G.; Diamantoglou, S.; Tajmir-Riahi, H. A.

    2006-10-01

    Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. Flavonoids are powerful antioxidants and prevent DNA damage. The antioxidative protections are related to their binding modes to DNA duplex and complexation with free radicals in vivo. However, flavonoids are known to inhibit the activities of several enzymes such as calcium phospholipid-dependent protein kinase, tyrosine protein kinase from rat lung, phosphorylase kinase, phosphatidylinositol 3-kinase and DNA topoisomerases that exhibit the importance of flavonoid-protein interaction. This study was designed to examine the interaction of human serum albumin (HSA) with quercetin (que), kaempferol (kae) and delphinidin (del) in aqueous solution at physiological conditions, using constant protein concentration of 0.25 mM (final) and various drug contents of 1 μM-1 mM. FTIR and UV-vis spectroscopic methods were used to determine the polyphenolic binding mode, the binding constant and the effects of flavonoid complexation on protein secondary structure. The spectroscopic results showed that flavonoids are located along the polypeptide chains through H-bonding interactions with overall affinity constant of Kque = 1.4 × 10 4 M -1, Kkae = 2.6 × 10 5 M -1 and Kdel = 4.71 × 10 5 M -1. The protein secondary structure showed no alterations at low pigment concentration (1 μM), whereas at high flavonoid content (1 mM), major reduction of α-helix from 55% (free HSA) to 42-46% and increase of β-sheet from 15% (free HSA) to 17-19% and β-anti from 7% (free HSA) to 10-20% occurred in the flavonoid-HSA adducts. The major reduction of HSA α-helix is indicative of a partial protein unfolding upon flavonoid interaction.

  11. BMP-2 Overexpression Augments Vascular Smooth Muscle Cell Motility by Upregulating Myosin Va via Erk Signaling

    Directory of Open Access Journals (Sweden)

    Ming Zhang

    2014-01-01

    Full Text Available Background. The disruption of physiologic vascular smooth muscle cell (VSMC migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2. Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy.

  12. Small GTP-binding proteins in human endothelial cells

    NARCIS (Netherlands)

    de Leeuw, H. P.; Koster, P. M.; Calafat, J.; Janssen, H.; van Zonneveld, A. J.; van Mourik, J. A.; Voorberg, J.

    1998-01-01

    Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of

  13. Raman spectroscopy in investigations of mechanism of binding of human serum albumin to molecular probe fluorescein

    International Nuclear Information System (INIS)

    Vlasova, I M; Saletsky, A M

    2008-01-01

    The mechanism of binding of molecular probe fluorescein to molecules of human serum albumin was studied by the Raman spectroscopy method. The position of binding Center on human serum albumin molecule for fluorescein is determined. The amino acid residues of albumin molecule, participating in binding of fluorescein at different pH values of solution, are established. The conformation rearrangements of globules of human serum albumin, taking place at binding of fluorescein at different pH values of solution, are registered

  14. Muscle protein analysis. II. Two-dimensional electrophoresis of normal and diseased human skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Giometti, C.S. (Argonne National Lab., IL); Barany, M.; Danon, M.J.; Anderson, N.G.

    1980-07-01

    High-resolution two-dimensional electrophoresis was used to analyze the major proteins of normal and pathological human-muscle samples. The normal human-muscle pattern contains four myosin light chains: three that co-migrate with the myosin light chains from rabbit fast muscle (extensor digitorum longus), and one that co-migrates with the light chain 2 from rabbit slow muscle (soleus). Of seven Duchenne muscular dystrophy samples, four yielded patterns with decreased amounts of actin and myosin relative to normal muscle, while three samples gave patterns comparable to that for normal muscle. Six samples from patients with myotonic dystrophy also gave normal patterns. In nemaline rod myopathy, in contrast, the pattern was deficient in two of the fast-type myosin light chains.

  15. Distinct p53 genomic binding patterns in normal and cancer-derived human cells

    Energy Technology Data Exchange (ETDEWEB)

    Botcheva K.; McCorkle S. R.; McCombie W. R.; Dunn J. J.; Anderson C. W.

    2011-12-15

    We report here genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40% were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied, functional p53 binding sites and, to date, not observed by previous p53 genome-wide studies. Nearly half of the high-confidence binding sites in the IMR90 cells reside in CpG islands, in marked contrast to sites reported in cancer-derived cells. The distinct genomic features of the IMR90 binding sites do not reflect a distinct preference for specific sequences, since the de novo developed p53 motif based on our study is similar to those reported by genome-wide studies of cancer cells. More likely, the different chromatin landscape in normal, compared with cancer-derived cells, influences p53 binding via modulating availability of the sites. We compared the IMR90 ChIPseq peaks to the recently published IMR90 methylome1 and demonstrated that they are enriched at hypomethylated DNA. Our study represents the first genome-wide, de novo mapping of p53 binding sites in normal human cells and reveals that p53 binding sites reside in distinct genomic landscapes in normal and cancer-derived human cells.

  16. Human faces are slower than chimpanzee faces.

    Directory of Open Access Journals (Sweden)

    Anne M Burrows

    Full Text Available While humans (like other primates communicate with facial expressions, the evolution of speech added a new function to the facial muscles (facial expression muscles. The evolution of speech required the development of a coordinated action between visual (movement of the lips and auditory signals in a rhythmic fashion to produce "visemes" (visual movements of the lips that correspond to specific sounds. Visemes depend upon facial muscles to regulate shape of the lips, which themselves act as speech articulators. This movement necessitates a more controlled, sustained muscle contraction than that produced during spontaneous facial expressions which occur rapidly and last only a short period of time. Recently, it was found that human tongue musculature contains a higher proportion of slow-twitch myosin fibers than in rhesus macaques, which is related to the slower, more controlled movements of the human tongue in the production of speech. Are there similar unique, evolutionary physiologic biases found in human facial musculature related to the evolution of speech?Using myosin immunohistochemistry, we tested the hypothesis that human facial musculature has a higher percentage of slow-twitch myosin fibers relative to chimpanzees (Pan troglodytes and rhesus macaques (Macaca mulatta. We sampled the orbicularis oris and zygomaticus major muscles from three cadavers of each species and compared proportions of fiber-types. Results confirmed our hypothesis: humans had the highest proportion of slow-twitch myosin fibers while chimpanzees had the highest proportion of fast-twitch fibers.These findings demonstrate that the human face is slower than that of rhesus macaques and our closest living relative, the chimpanzee. They also support the assertion that human facial musculature and speech co-evolved. Further, these results suggest a unique set of evolutionary selective pressures on human facial musculature to slow down while the function of this muscle

  17. SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

    Science.gov (United States)

    Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

    2016-04-01

    Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

  18. Comparative studies of human and chicken retinol-binding proteins and prealbumins.

    Science.gov (United States)

    Kopelman, M; Mokady, S; Cogan, U

    1976-08-09

    Microheterogeneity of retinol-binding proteins of human plasma and urine, and of chicken plasma was studied by polyacrylamide gel electrophoresis. All three protein systems were found microheterogenous. Incorporation of retinol into the protein preparations on the one hand, and depletion of these proteins from retinol on the other hand, enabled us to clarify the extent to which the presence or absence of the ligand affects the apparent heterogeneity. Upon electrophoresis, each of the native proteins displayed two pairs of protein zones. It appeared that within each pair the fast moving band corresponded to aporetinol-binding protein which upon binding of retinol was converted to a holoprotein with a slightly lower mobility. However, it did not seem that proteins of one pair were converted to proteins of the second pair upon binding of retinol, substantiating ghe microheterogenous character of this protein system. A rapid, two step procedure for isolation of prealbumins from plasma is described. The method which consists of DEAE-cellulose chromatography follwed by preparative electrophoresis was utilized to separate human and chicken prealbumins. Routine dodecyl sulphate electrophoresis resulted in partial dissociation of human prealbumin but in no dissociation of the chicken protein. More drastic treatments prior to electrophoresis were needed to effect complete disruption of both proteins into subunits.

  19. Myosin light chain phosphorylation is critical for adaptation to cardiac stress.

    Science.gov (United States)

    Warren, Sonisha A; Briggs, Laura E; Zeng, Huadong; Chuang, Joyce; Chang, Eileen I; Terada, Ryota; Li, Moyi; Swanson, Maurice S; Lecker, Stewart H; Willis, Monte S; Spinale, Francis G; Maupin-Furlowe, Julie; McMullen, Julie R; Moss, Richard L; Kasahara, Hideko

    2012-11-27

    Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in the adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac myosin light-chain kinase (cMLCK), yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined. We found that pressure overload induced by transaortic constriction in wild-type mice reduced phosphorylated MLC2v levels by ≈40% and cMLCK levels by ≈85%. To examine how a reduction in cMLCK and the corresponding reduction in phosphorylated MLC2v affect function, we generated Mylk3 gene-targeted mice and transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure overload led to severe heart failure in cMLCK knockout mice but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems. Our results suggest the novel idea that accelerated cMLCK protein turnover by the ubiquitin-proteasome system underlies the transition from compensated hypertrophy to decompensated heart failure as a result of reduced phosphorylation of MLC2v.

  20. Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

    Science.gov (United States)

    Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

    2018-05-15

    All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret

  1. Myosin XI-dependent formation of tubular structures from endoplasmic reticulum isolated from tobacco cultured BY-2 cells.

    Science.gov (United States)

    Yokota, Etsuo; Ueda, Haruko; Hashimoto, Kohsuke; Orii, Hidefumi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Shimmen, Teruo

    2011-05-01

    The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER.

  2. Polar bear hemoglobin and human Hb A0: same 2,3-diphosphoglycerate binding site but asymmetry of the binding?

    Science.gov (United States)

    Pomponi, Massimo; Bertonati, Claudia; Patamia, Maria; Marta, Maurizio; Derocher, Andrew E; Lydersen, Christian; Kovacs, Kit M; Wiig, Oystein; Bårdgard, Astrid J

    2002-11-01

    Polar bear (Ursus maritimus) hemoglobin (Hb) shows a low response to 2,3-diphosphoglycerate (2,3-DPG), compared to human Hb A0, even though these proteins have the same 2,3-DPG-binding site. In addition, polar bear Hb shows a high response to chloride and an alkaline Bohr effect (deltalog P50/deltapH) that is significantly greater than that of human Hb A0. The difference in sequence Pro (Hb A0)-->Gly (polar bear Hb) at position A2 in the A helix seems to be critical for reduced binding of 2,3-DPG. Our results also show that the A2 position may influence not only the flexibility of the A helix, but that differences in flexibility of the first turn of the A helix may affect the unloading of oxygen for the intrinsic ligand affinities of the alpha and beta chains. However, preferential binding to either chain can only take place if there is appreciable asymmetric binding of the phosphoric effector. Regarding this point, 31P NMR data suggest a loss of symmetry of the 2,3-DPG-binding site in the deoxyHb-2,3-DPG complex.

  3. Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies

    OpenAIRE

    Payne, Ruth O.; Silk, Sarah E.; Elias, Sean C.; Milne, Kathryn H.; Rawlinson, Thomas A.; Llewellyn, David; Shakri, A. Rushdi; Jin, Jing; Labb?, Genevi?ve M.; Edwards, Nick J.; Poulton, Ian D.; Roberts, Rachel; Farid, Ryan; J?rgensen, Thomas; Alanine, Daniel G.W.

    2017-01-01

    BACKGROUND: Plasmodium vivax is the most widespread human malaria geographically; however, no effective vaccine exists. Red blood cell invasion by the P. vivax merozoite depends on an interaction between the Duffy antigen receptor for chemokines (DARC) and region II of the parasite's Duffy-binding protein (PvDBP_RII). Naturally acquired binding-inhibitory antibodies against this interaction associate with clinical immunity, but it is unknown whether these responses can be induced by human vac...

  4. Insulin binding properties of normal and transformed human epidermal cultured keratinocytes

    International Nuclear Information System (INIS)

    Verrando, P.; Ortonne, J.P.

    1985-01-01

    Insulin binding to its receptors was studied in cultured normal and transformed (A431 line) human epidermal keratinocytes. The specific binding was a temperature-dependent, saturable process. Normal keratinocytes possess a mean value of about 80,000 receptors per cell. Fifteen hours exposure of the cells to insulin lowered their receptor number (about 65% loss in available sites); these reappeared when the hormone was removed from the culture medium. In the A431 epidermoid carcinoma cell line, there is a net decrease in insulin binding (84% of the initial bound/free hormone ratio in comparison with normal cells) essentially related to a loss in receptor affinity for insulin. Thus, cultured human keratinocytes which express insulin receptors may be a useful tool in understanding skin pathology related to insulin disorders

  5. The local expression of adult chicken heart myosins during development. I. The three days embryonic chicken heart

    NARCIS (Netherlands)

    Sanders, E.; Moorman, A. F.; Los, J. A.

    1984-01-01

    Immunofluorescence studies were performed on serial sections of three days embryonic chicken hearts using antibodies specific for adult atrial and ventricular myosin heavy chains respectively. The anti-ventricular myosin serum reacted with the entire myocardium showing a decreasing intensity going

  6. CALIX[4]ARENE C-99 INHIBITS MYOSIN ATPase ACTIVITY AND CHANGES THE ORGANIZATION OF CONTRACTILE FILAMENTS OF MYOMETRIUM.

    Science.gov (United States)

    Labyntseva, R D; Bevza, A A; Lul'ko, A O; Cherenok, S O; Kalchenko, V I; Kosterin, S O

    2015-01-01

    Calix[4]arenes are cup-like macrocyclic (polyphenolic) compounds, they are regarded as promising molecular "platforms" for the design of new physiologically active compounds. We have earlier found that calix[4]arene C-99 inhibits the ATPase activity of actomyosin and myosin subfragment-1 of pig uterus in vitro. The aim of this study was to investigate the interaction of calix[4]arene C-99 with myosin from rat uterine myocytes. It was found that the ATPase activity of myosin prepared from pre-incubated with 100 mM of calix[4]arene C-99 myocytes was almost 50% lower than in control. Additionally, we have revealed the effect of calix[4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy. This effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix[4]arene. The obtained results demonstrate the ability of calix[4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin ATPase activity, but also the structure of the actin and myosin filaments in the myometrial cells. Demonstrated ability of calix[4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.

  7. Calix[4]arene C-99 inhibits myosin ATPase activity and changes the organization of contractile filaments of myometrium

    Directory of Open Access Journals (Sweden)

    R. D. Labyntseva,

    2015-12-01

    Full Text Available Calix[4]arenes are cup-like macrocyclic (polyphenolic compounds, they are regarded as promising molecular “platforms” for the design of new physiologically active compounds. We have earlier found that сalix[4]arenе C-99 inhibits the ATPase activity of actomyosin and myosin subfragment-1 of pig uterus іn vitro. The aim of this study was to investigate the interaction of calix[4]arene C-99 with myosin from rat uterine myocytes. It was found that the ATPase activity of myosin prepared from pre-incubated with 100 mM of calix[4]arene C-99 myocytes was almost 50% lower than in control. Additionally, we have revealed the effect of calix[4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy. This effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix[4]arene. The obtained results demonstrate the ability of calix[4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin ATPase activity, but also the structure of the actin and myosin filaments in the myometrial cells. Demonstrated ability of calix[4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.

  8. Specific binding of lactoferrin to Escherichia coli isolated from human intestinal infections

    International Nuclear Information System (INIS)

    Naidu, S.S.; Erdei, J.; Forsgren, A.; Naidu, A.S.; Czirok, E.; Gado, I.; Kalfas, S.; Thoren, A.

    1991-01-01

    The degrees of human lactoferrin (HLf) and bovine lactoferrin (BLf) binding in 169 Escherichia coli strains isolated from human intestinal infections, and in an additional 68 strains isolated from healthy individuals, were examined in a 125 I-labelled protein binding assay. The binding was expressed as a percentage calculated from the total labelled ligand added to bacteria. The HLf and BLf binding to E. coli was in the range 3.7 to 73.4% and 4.8 to 61.6%, respectively. Enterotoxigenic strains demonstrated a significantly higher HLf binding (median = 19%) than enteropathogenic, enteroinvasive, enterohaemorrhagic strains or normal intestinal E. coli isolates (medians 6 to 9). Enteropathogenic strains belonging to serotypes O44 and O127 demonstrated significantly higher HLf binding compared to O26, O55, O111, O119 and O126. No significant differences in the degree of HLf or BLf binding were found between aerobactin-producing and non-producing strains. The interaction was further characterized in a high Lf-binging EPEC strain, E34663 (serotype O127). The binding was stable in the pH range 4.0 to 7.5, did not dissociate in the presence of 2M NaCl or 2M urea, and reached saturation within two h. Unlabelled HLf and BLf displaced the 125 I-HLf binding to E34663 in a dose-dependent manner. Apo- and iron-saturated forms of Lf demonstrated similar binding to E34663. Among various unlabelled subephithelial matrix proteins and carbohydrates tested (in 10 4 -fold excess) only fibronectin and fibrinogen caused a moderate inhibition of 125 I-HLf binding. According to Scatchard plot analysis, 5,400 HLf-binding sites/cell, with an affinity constant (K a ) of 1.4 x 10 -7 M, were estimated in strain E34663. These data establish the presence of a specific Lf-binding mechanism in E. coli. (au)

  9. Clinical study on the time courses of serum myosin light chain I levels in patients with acute myocardial infarction

    International Nuclear Information System (INIS)

    Nakanishi, Masako; Saiki, Yasuhiko; Ui, Kazuyo

    1992-01-01

    Changes of serum myosin light chain I (Myosin LCI) concentrations and creatine kinase (CK) activities were serially measured in 23 patients with acute myocardial infarction. Intracoronary thrombolysis was performed in 14 patients (ICT group) while the remaining 9 patients were treated in the conventional manner (non ICT group). The relationships between the maximum levels of serum Myosin LCI or CK and a myocardial infarct size index or left ventricular function were evaluated in 18 patients. The myocardial infarct size index was determined by 201 Tl myocardial scintigrams performed in the chronic phase. Multiple peaks of Myosin LCI were observed in 64% (9/14) of the ICT group and the first peak in 6 of these patients appeared much earlier in the same time as CK peak than in the non-ICT group, while multiple peaks were seen only in one case in the non-ICT group. The infarct size index by 201 Tl myocardial SPECT correlated with maximum Myosin LCI levels (r=0.88, p<0.001, n=10) and CK activities (r=0.67, p<0.05, n=10). These results indicate that the measurement of serum Myosin LCI is very useful for estimating the extent of myocardial damage and suggest that myocardial degeneration occurs at a very early phase of myocardial infarction. (author)

  10. Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography

    International Nuclear Information System (INIS)

    Gates, T.S.; Zimmerman, R.P.; Mantyh, C.R.; Vigna, S.R.; Maggio, J.E.; Welton, M.L.; Passaro, E.P. Jr.; Mantyh, P.W.

    1988-01-01

    Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for 125 I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: (1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; (2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; (3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and (4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells

  11. Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion

    Energy Technology Data Exchange (ETDEWEB)

    Wasik, Anita A.; Dumont, Vincent [Department of Pathology, University of Helsinki, 00014 Helsinki (Finland); Tienari, Jukka [Department of Pathology, University of Helsinki and Helsinki University Hospital, 00290 Helsinki, 05850 Hyvinkää (Finland); Nyman, Tuula A. [Institute of Biotechnology, University of Helsinki, 00014 Helsinki (Finland); Fogarty, Christopher L.; Forsblom, Carol; Lehto, Markku [Folkhälsan Institute of Genetics, Folkhälsan Research Center, 00290 Helsinki (Finland); Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki (Finland); Diabetes& Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki (Finland); Lehtonen, Eero [Department of Pathology, University of Helsinki, 00014 Helsinki (Finland); Laboratory Animal Centre, University of Helsinki, 00014 Helsinki (Finland); Groop, Per-Henrik [Folkhälsan Institute of Genetics, Folkhälsan Research Center, 00290 Helsinki (Finland); Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki (Finland); Diabetes& Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki (Finland); Baker IDI Heart & Diabetes Institute, 3004 Melbourne (Australia); Lehtonen, Sanna, E-mail: sanna.h.lehtonen@helsinki.fi [Department of Pathology, University of Helsinki, 00014 Helsinki (Finland)

    2017-01-15

    Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane. - Highlights: • Septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA) and SNAP23 form a complex. • Knockdown of septin 7 increases NM-IIA activity in the SNAP23 complex. • Insulin decreases septin 7 level and increases NM-IIA activity in the SNAP23 complex. • Septin 7 hinders GSV docking/fusion by reducing NM-IIA activity in the SNAP23 complex.

  12. Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion

    International Nuclear Information System (INIS)

    Wasik, Anita A.; Dumont, Vincent; Tienari, Jukka; Nyman, Tuula A.; Fogarty, Christopher L.; Forsblom, Carol; Lehto, Markku; Lehtonen, Eero; Groop, Per-Henrik; Lehtonen, Sanna

    2017-01-01

    Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane. - Highlights: • Septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA) and SNAP23 form a complex. • Knockdown of septin 7 increases NM-IIA activity in the SNAP23 complex. • Insulin decreases septin 7 level and increases NM-IIA activity in the SNAP23 complex. • Septin 7 hinders GSV docking/fusion by reducing NM-IIA activity in the SNAP23 complex.

  13. Proteomic analysis of heparin-binding proteins from human seminal ...

    Indian Academy of Sciences (India)

    Prakash

    (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 ... Thus, the combined effects of seminal plasma components support the survival of ...... The BBXB motif of RANTES is the principal site for heparin binding and controls ...

  14. Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Ayaho; Kanaba, Teppei [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan); Satoh, Ryosuke [Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku 141-0021, Tokyo (Japan); Fujiwara, Toshinobu [Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku 141-0021, Tokyo (Japan); Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku,Nagoya 467-8603 (Japan); Ito, Yutaka [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan); Sugiura, Reiko [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan); Mishima, Masaki, E-mail: mishima-masaki@tmu.ac.jp [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan)

    2013-07-19

    Highlights: •Solution structure of the second RRM of Nrd1 was determined. •RNA binding site of the second RRM was estimated. •Regulatory mechanism of RNA binding by phosphorylation is discussed. -- Abstract: Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.

  15. Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation

    International Nuclear Information System (INIS)

    Kobayashi, Ayaho; Kanaba, Teppei; Satoh, Ryosuke; Fujiwara, Toshinobu; Ito, Yutaka; Sugiura, Reiko; Mishima, Masaki

    2013-01-01

    Highlights: •Solution structure of the second RRM of Nrd1 was determined. •RNA binding site of the second RRM was estimated. •Regulatory mechanism of RNA binding by phosphorylation is discussed. -- Abstract: Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed

  16. Differential recognition of obligate anaerobic bacteria by human mannose-binding lectin.

    Science.gov (United States)

    Townsend, R; Read, R C; Turner, M W; Klein, N J; Jack, D L

    2001-05-01

    Deficiency of the innate, humoral immune component mannose-binding lectin (MBL) predisposes individuals to a variety of infections, but the importance of MBL in infection by anaerobes has not been addressed. The attachment of MBL to a wide range of anaerobic bacteria associated with human disease and colonization was surveyed. The results suggest that for the species we examined, resistance to MBL binding may be associated with organisms that are more commonly pathogenic and that MBL binding to some bacteria may be phase variable.

  17. Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Melkani, Girish C.; Lee, Chi F.; Cammarato, Anthony [Department of Biology and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614 (United States); Bernstein, Sanford I., E-mail: sbernst@sciences.sdsu.edu [Department of Biology and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614 (United States)

    2010-05-28

    UNC-45 belongs to the UCS (UNC-45, CRO1, She4p) domain protein family, whose members interact with various classes of myosin. Here we provide structural and biochemical evidence that Escherichia coli-expressed Drosophila UNC-45 (DUNC-45) maintains the integrity of several substrates during heat-induced stress in vitro. DUNC-45 displays chaperone function in suppressing aggregation of the muscle myosin heavy meromyosin fragment, the myosin S-1 motor domain, {alpha}-lactalbumin and citrate synthase. Biochemical evidence is supported by electron microscopy, which reveals the first structural evidence that DUNC-45 prevents inter- or intra-molecular aggregates of skeletal muscle heavy meromyosin caused by elevated temperatures. We also demonstrate for the first time that UNC-45 is able to refold a denatured substrate, urea-unfolded citrate synthase. Overall, this in vitro study provides insight into the fate of muscle myosin under stress conditions and suggests that UNC-45 protects and maintains the contractile machinery during in vivo stress.

  18. Platelet binding and biodistribution of [99mTc]rBitistatin in animal species and humans

    International Nuclear Information System (INIS)

    Knight, Linda C.; Romano, Jan E.; Bright, Lewis T.; Agelan, Alexis; Kantor, Steven; Maurer, Alan H.

    2007-01-01

    Introduction: 99m Tc recombinant bitistatin (rBitistatin) is a radioligand for α IIb β 3 (glycoproteins IIb/IIIa) receptor on platelets and is being developed as a diagnostic radiopharmaceutical for in vivo imaging of acute thrombi and emboli. Prior to the first administration of [ 99m Tc]rBitistatin to human subjects, its biodistribution and effects on platelets were evaluated in animals. This paper reports findings in animal studies in comparison with initial findings in normal human subjects. Methods: [ 99m Tc]rBitistatin was administered to mice, guinea pigs and dogs to assess time-dependent organ distribution, urinary excretion and blood disappearance rates. Blood samples were analyzed to determine radioligand binding to circulating platelets and the extent of plasma protein binding. The effect of [ 99m Tc]rBitistatin on circulating platelet count was determined. These factors were also determined in normal human subjects who received [ 99m Tc]rBitistatin as part of a Phase I clinical trial. Results: The main organs that accumulated [ 99m Tc]rBitistatin were kidneys, liver and spleen in all animal species and humans. The main organs seen on human images were the kidneys and spleen. Liver uptake was fainter, and soft-tissue background was low. [ 99m Tc]rBitistatin bound to circulating platelets in blood, with a higher percentage of binding to platelets in guinea pigs and dogs compared to that in humans. Plasma protein binding was low and of little consequence in view of platelet binding. The main route of excretion was through the urine. [ 99m Tc]rBitistatin did not affect platelet counts in humans or dogs. Conclusions: [ 99m Tc]rBitistatin, when administered at low doses for imaging, has no adverse effects on platelets and has the qualitative biodistribution predicted by animal studies. [ 99m Tc]rBitistatin was found to bind to circulating platelets in humans, suggesting that it will be able to bind to activated platelets in vivo in patients with acute

  19. Blebbistatin, a myosin II inhibitor, suppresses Ca(2+)-induced and "sensitized"-contraction of skinned tracheal muscles from guinea pig.

    Science.gov (United States)

    Yumoto, Masatoshi; Watanabe, Masaru

    2013-01-01

    Blebbistatin, a potent inhibitor of myosin II, has inhibiting effects on Ca(2+)-induced contraction and contractile filament organization without affecting the Ca(2+)-sensitivity to the force and phosphorylation level of myosin regulatory light chain (MLC20) in skinned (cell membrane permeabilized) taenia cecum from the guinea pig (Watanabe et al., Am J Physiol Cell Physiol. 2010; 298: C1118-26). In the present study, we investigated blebbistatin effects on the contractile force of skinned tracheal muscle, in which myosin filaments organization is more labile than that in the taenia cecum. Blebbistatin at 10 μM or higher suppressed Ca(2+)-induced tension development at any given Ca(2+) concentration, but had little effects on the Ca(2+)- induced myosin light chain phosphorylation. Also blebbistatin at 10 μM and higher significantly suppressed GTP-γS-induced "sensitized" force development. Since the force inhibiting effects of blebbistatin on the skinned trachea were much stronger than those in skinned taenia cecum, blebbistatin might directly affect myosin filaments organization.

  20. Lignans from the roots of Urtica dioica and their metabolites bind to human sex hormone binding globulin (SHBG).

    Science.gov (United States)

    Schöttner, M; Gansser, D; Spiteller, G

    1997-12-01

    Polar extracts of the stinging nettle (Urtica dioica L.) roots contain the ligans (+)-neoolivil, (-)-secoisolariciresinol, dehydrodiconiferyl alcohol, isolariciresinol, pinoresinol, and 3,4-divanillyltetrahydrofuran. These compounds were either isolated from Urtica roots, or obtained semisynthetically. Their affinity to human sex hormone binding globulin (SHBG) was tested in an in vitro assay. In addition, the main intestinal transformation products of plant lignans in humans, enterodiol and enterolactone, together with enterofuran were checked for their activity. All lignans except (-)-pinoresinol developed a binding affinity to SHBG in the in vitro assay. The affinity of (-)-3,4-divanillyltetrahydrofuran was outstandingly high. These findings are discussed with respect to potential beneficial effects of plant lignans on benign prostatic hyperplasia (BPH).

  1. The Rho kinases I and II regulate different aspects of myosin II activity

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Multhaupt, Hinke A B; Couchman, John R

    2005-01-01

    The homologous mammalian rho kinases (ROCK I and II) are assumed to be functionally redundant, based largely on kinase construct overexpression. As downstream effectors of Rho GTPases, their major substrates are myosin light chain and myosin phosphatase. Both kinases are implicated in microfilament...... bundle assembly and smooth muscle contractility. Here, analysis of fibroblast adhesion to fibronectin revealed that although ROCK II was more abundant, its activity was always lower than ROCK I. Specific reduction of ROCK I by siRNA resulted in loss of stress fibers and focal adhesions, despite...

  2. Effects of BTS (N-benzyl-p-toluene sulphonamide), an inhibitor for myosin-actin interaction, on myofibrillogenesis in skeletal muscle cells in culture.

    Science.gov (United States)

    Kagawa, Maiko; Sato, Naruki; Obinata, Takashi

    2006-11-01

    Actin filaments align around myosin filaments in the correct polarity and in a hexagonal arrangement to form cross-striated structures. It has been postulated that this myosin-actin interaction is important in the initial phase of myofibrillogenesis. It was previously demonstrated that an inhibitor of actin-myosin interaction, BDM (2,3-butanedione monoxime), suppresses myofibril formation in muscle cells in culture. However, further study showed that BDM also exerts several additional effects on living cells. In this study, we further examined the role of actin-myosin interaction in myofibril assembly in primary cultures of chick embryonic skeletal muscle by applying a more specific inhibitor, BTS (N-benzyl-p-toluene sulphonamide), of myosin ATPase and actin-myosin interaction. The assembly of sarcomeric structures from myofibrillar proteins was examined by immunocytochemical methods with the application of BTS to myotubes just after fusion. Addition of BTS (10-50 microM) significantly suppressed the organization of actin and myosin into cross-striated structures. BTS also interfered in the organization of alpha-actinin, C-protein (or MyBP-C), and connectin (or titin) into ordered striated structures, though the sensitivity was less. Moreover, when myotubes cultured in the presence of BTS were transferred to a control medium, sarcomeric structures were formed in 2-3 days, indicating that the inhibitory effect of BTS on myotubes is reversible. These results show that actin-myosin interaction plays a critical role in the process of myofibrillogenesis.

  3. Discoidin Domain Receptor 1 Mediates Myosin-Dependent Collagen Contraction

    Directory of Open Access Journals (Sweden)

    Nuno M. Coelho

    2017-02-01

    Full Text Available Discoidin domain receptor 1 (DDR1 is a tyrosine kinase collagen adhesion receptor that mediates cell migration through association with non-muscle myosin IIA (NMIIA. Because DDR1 is implicated in cancer fibrosis, we hypothesized that DDR1 interacts with NMIIA to enable collagen compaction by traction forces. Mechanical splinting of rat dermal wounds increased DDR1 expression and collagen alignment. In periodontal ligament of DDR1 knockout mice, collagen mechanical reorganization was reduced >30%. Similarly, cultured cells with DDR1 knockdown or expressing kinase-deficient DDR1d showed 50% reduction of aligned collagen. Tractional remodeling of collagen was dependent on DDR1 clustering, activation, and interaction of the DDR1 C-terminal kinase domain with NMIIA filaments. Collagen remodeling by traction forces, DDR1 tyrosine phosphorylation, and myosin light chain phosphorylation were increased on stiff versus soft substrates. Thus, DDR1 clustering, activation, and interaction with NMIIA filaments enhance the collagen tractional remodeling that is important for collagen compaction in fibrosis.

  4. Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity.

    Science.gov (United States)

    Srinivasan, Sundaramoorthy; Fernández-Sampedro, Miguel A; Morillo, Margarita; Ramon, Eva; Jiménez-Rosés, Mireia; Cordomí, Arnau; Garriga, Pere

    2018-03-27

    Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. Probing muscle myosin motor action: x-ray (m3 and m6) interference measurements report motor domain not lever arm movement.

    Science.gov (United States)

    Knupp, Carlo; Offer, Gerald; Ranatunga, K W; Squire, John M

    2009-07-10

    The key question in understanding how force and movement are produced in muscle concerns the nature of the cyclic interaction of myosin molecules with actin filaments. The lever arm of the globular head of each myosin molecule is thought in some way to swing axially on the actin-attached motor domain, thus propelling the actin filament past the myosin filament. Recent X-ray diffraction studies of vertebrate muscle, especially those involving the analysis of interference effects between myosin head arrays in the two halves of the thick filaments, have been claimed to prove that the lever arm moves at the same time as the sliding of actin and myosin filaments in response to muscle length or force steps. It was suggested that the sliding of myosin and actin filaments, the level of force produced and the lever arm angle are all directly coupled and that other models of lever arm movement will not fit the X-ray data. Here, we show that, in addition to interference across the A-band, which must be occurring, the observed meridional M3 and M6 X-ray intensity changes can all be explained very well by the changing diffraction effects during filament sliding caused by heads stereospecifically attached to actin moving axially relative to a population of detached or non-stereospecifically attached heads that remain fixed in position relative to the myosin filament backbone. Crucially, and contrary to previous interpretations, the X-ray interference results provide little direct information about the position of the myosin head lever arm; they are, in fact, reporting relative motor domain movements. The implications of the new interpretation are briefly assessed.

  6. Ulex europaeus I and glycine max bind to the human olfactory bulb.

    Science.gov (United States)

    Nagao, M; Oka, N; Kamo, H; Akiguchi, I; Kimura, J

    1993-12-24

    The distribution of binding sites for the fucose-selective lectin Ulex europaeus I and the terminal N-acetylgalactosamine-selective lectin glycine max in the human olfactory bulb were studied. These lectins bound to primary olfactory axons in the olfactory nerve layer and the glomerular layer. They also bound to fibers located in the deeper layers such as the external plexiform layer and the granular layer. Furthermore they projected to the olfactory stalk but not in the cerebrum. The deeper projections of the lectin binding fibers may affect the function of the olfactory bulb in humans.

  7. Myosin Va Plays a Role in Nitrergic Smooth Muscle Relaxation in Gastric Fundus and Corpora Cavernosa of Penis

    Science.gov (United States)

    Carew, Josephine A.; Goyal, Raj K.; Sullivan, Maryrose P.

    2014-01-01

    The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction. PMID:24516539

  8. Effects of human low and high density lipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes

    International Nuclear Information System (INIS)

    Brissette, L.; Nol, S.P.

    1986-01-01

    Upon incubation with rat liver membranes, radioiodinated rat intermediate density lipoproteins (IDL) interacted with at least two binding sites having a low and a high affinity as demonstrated by the curvilinear Scatchard plots obtained from the specific binding data. The purpose of our work was to identify the nature of these binding sites. Human low density lipoproteins (LDL), contain apolipoprotein B only, and human high density lipoproteins (HDL3), containing neither apolipoprotein B nor E, were both capable of decreasing the specific binding of rat 125 I-IDL. The Scatchard analysis clearly revealed that only the low affinity component was affected by the addition of these human lipoproteins. In fact, the low affinity binding component gradually decreased as the amount of human LDL or HDL3 increased in the binding assay. At a 200-fold excess of human LDL or HDL3, the low affinity binding was totally masked, and the Scatchard plot of the specific 125 I-IDL binding became linear. Only the high affinity binding component was left, enabling a precise measurement of its binding parameters. In a series of competitive displacement experiments in which the binding assay contained a 200-fold excess of human LDL or HDL3, only unlabeled rat IDL effectively displaced the binding of rat 125 I-IDL. We conclude that the low affinity binding of rat IDL to rat liver membranes is due to weak interactions with unspecified lipoprotein binding sites. The camouflage of these sites by human lipoproteins makes possible the study of IDL binding to the high affinity component which likely represents the combined effect of IDL binding to both the remnant and the LDL receptors

  9. Carboxyl-terminal-dependent recruitment of nonmuscle myosin II to megakaryocyte contractile ring during polyploidization.

    Science.gov (United States)

    Badirou, Idinath; Pan, Jiajia; Legrand, Céline; Wang, Aibing; Lordier, Larissa; Boukour, Siham; Roy, Anita; Vainchenker, William; Chang, Yunhua

    2014-10-16

    Endomitosis is a unique megakaryocyte (MK) differentiation process that is the consequence of a late cytokinesis failure associated with a contractile ring defect. Evidence from in vitro studies has revealed the distinct roles of 2 nonmuscle myosin IIs (NMIIs) on MK endomitosis: only NMII-B (MYH10), but not NMII-A (MYH9), is localized in the MK contractile ring and implicated in mitosis/endomitosis transition. Here, we studied 2 transgenic mouse models in which nonmuscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. This study provides in vivo evidence on the specific role of NMII-B on MK polyploidization. It demonstrates that the carboxyl-terminal domain of the heavy chains determines myosin II localization to the MK contractile ring and is responsible for the specific role of NMII-B in MK polyploidization.

  10. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  11. A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors

    Energy Technology Data Exchange (ETDEWEB)

    de Vries, Robert P.; Tzarum, Netanel; Peng, Wenjie; Thompson, Andrew J.; Ambepitiya Wickramasinghe, Iresha N.; de la Pena, Alba T. Torrents; van Breemen, Marielle J.; Bouwman, Kim M.; Zhu, Xueyong; McBride, Ryan; Yu, Wenli; Sanders, Rogier W.; Verheije, Monique H.; Wilson, Ian A.; Paulson, James C.

    2017-07-10

    In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding.

  12. Alterations in rat cardiac myosin isozymes induced by whole-body irradiation are prevented by 3,5,3'-L-triiodothyronine

    International Nuclear Information System (INIS)

    Litten, R.Z.; Fein, H.G.; Gainey, G.T.; Walden, T.L.; Smallridge, R.C.

    1990-01-01

    Changes in cardiac myosin isozymes and serum thyroid hormone levels were investigated in rats following 10 Gy whole-body gamma irradiation. The percent beta-myosin heavy chain increased from 21.3 ± 1.8 to 28.1 ± 6.8 (NS) at 3-day postirradiation, 37.7 ± 1.9 (P less than .001) at 6-day postirradiation, and 43.8 ± 3.3 (P less than .001) at 9-day postirradiation. Along with the change in myosin isozymes was a significant 53% decrease (P less than .001) in the serum thyroxine (T4) level by day 3 postirradiation, remaining depressed through day 9 postirradiation. The serum 3,5,3'-triiodothyronine (T3) level, however, was normal until day 9, when significant depression was also observed. In contrast, the thyroid-stimulating hormone (TSH) level was significantly increased by fourfold at day 3, returning to near normal values by day 9 postirradiation. Daily injections of physiological doses of T3 (0.3 microgram/100 g body weight) prevented the change in the myosin isozymes following whole-body irradiation. Daily pharmacological injections of T3 (3.0 micrograms/100 g body weight) to the irradiated rats produced a further decrease in the percent beta-myosin heavy chain (below control values) indicating tissue hyperthyroidism. Thus, this study suggests that the change in myosin isozymes following whole-body irradiation is caused by an alteration in thyroid hormone activity

  13. Spectroscopic characterization of furosemide binding to human carbonic anhydrase II.

    Science.gov (United States)

    Ranjbar, Samira; Ghobadi, Sirous; Khodarahmi, Reza; Nemati, Houshang

    2012-05-01

    This study reports the interaction between furosemide and human carbonic anhydrase II (hCA II) using fluorescence, UV-vis and circular dichroism (CD) spectroscopy. Fluorescence data indicated that furosemide quenches the intrinsic fluorescence of the enzyme via a static mechanism and hydrogen bonding and van der Walls interactions play the major role in the drug binding. The binding average distance between furosemide and hCA II was estimated on the basis of the theory of Förster energy transfer. Decrease of protein surface hydrophobicity was also documented upon furosemide binding. Chemical modification of hCA II using N-bromosuccinimide indicated decrease of the number of accessible tryptophans in the presence of furosemide. CD results suggested the occurance of some alterations in α-helical content as well as tertiary structure of hCA II upon drug binding. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Characterization of f-actin tryptophan phosphorescence in the presence and absence of tryptophan-free myosin motor domain.

    Science.gov (United States)

    Bódis, Emöke; Strambini, Giovanni B; Gonnelli, Margherita; Málnási-Csizmadia, András; Somogyi, Béla

    2004-08-01

    The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140-293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (tau(1) and tau(2)) and increasing by approximately 20% the longest component (tau(3)). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (tau(4), twice as long as tau(3)) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique.

  15. Ligand Binding Induces Conformational Changes in Human Cellular Retinol-binding Protein 1 (CRBP1) Revealed by Atomic Resolution Crystal Structures.

    Science.gov (United States)

    Silvaroli, Josie A; Arne, Jason M; Chelstowska, Sylwia; Kiser, Philip D; Banerjee, Surajit; Golczak, Marcin

    2016-04-15

    Important in regulating the uptake, storage, and metabolism of retinoids, cellular retinol-binding protein 1 (CRBP1) is essential for trafficking vitamin A through the cytoplasm. However, the molecular details of ligand uptake and targeted release by CRBP1 remain unclear. Here we report the first structure of CRBP1 in a ligand-free form as well as ultra-high resolution structures of this protein bound to either all-trans-retinol or retinylamine, the latter a therapeutic retinoid that prevents light-induced retinal degeneration. Superpositioning of human apo- and holo-CRBP1 revealed major differences within segments surrounding the entrance to the retinoid-binding site. These included α-helix II and hairpin turns between β-strands βC-βD and βE-βF as well as several side chains, such as Phe-57, Tyr-60, and Ile-77, that change their orientations to accommodate the ligand. Additionally, we mapped hydrogen bond networks inside the retinoid-binding cavity and demonstrated their significance for the ligand affinity. Analyses of the crystallographic B-factors indicated several regions with higher backbone mobility in the apoprotein that became more rigid upon retinoid binding. This conformational flexibility of human apo-CRBP1 facilitates interaction with the ligands, whereas the more rigid holoprotein structure protects the labile retinoid moiety during vitamin A transport. These findings suggest a mechanism of induced fit upon ligand binding by mammalian cellular retinol-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of LysosomesV⃞

    OpenAIRE

    Cordonnier, Marie-Neige; Dauzonne, Daniel; Louvard, Daniel; Coudrier, Evelyne

    2001-01-01

    An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed ...

  17. Platelet binding and biodistribution of [{sup 99m}Tc]rBitistatin in animal species and humans

    Energy Technology Data Exchange (ETDEWEB)

    Knight, Linda C. [Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140 (United States)], E-mail: lknight@temple.edu; Romano, Jan E. [Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140 (United States); Bright, Lewis T.; Agelan, Alexis [University Laboratory Animal Resources, Temple University School of Medicine, Philadelphia, PA 19140 (United States); Kantor, Steven; Maurer, Alan H. [Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140 (United States)

    2007-10-15

    Introduction: {sup 99m}Tc recombinant bitistatin (rBitistatin) is a radioligand for {alpha}{sub IIb}{beta}{sub 3} (glycoproteins IIb/IIIa) receptor on platelets and is being developed as a diagnostic radiopharmaceutical for in vivo imaging of acute thrombi and emboli. Prior to the first administration of [{sup 99m}Tc]rBitistatin to human subjects, its biodistribution and effects on platelets were evaluated in animals. This paper reports findings in animal studies in comparison with initial findings in normal human subjects. Methods: [{sup 99m}Tc]rBitistatin was administered to mice, guinea pigs and dogs to assess time-dependent organ distribution, urinary excretion and blood disappearance rates. Blood samples were analyzed to determine radioligand binding to circulating platelets and the extent of plasma protein binding. The effect of [{sup 99m}Tc]rBitistatin on circulating platelet count was determined. These factors were also determined in normal human subjects who received [{sup 99m}Tc]rBitistatin as part of a Phase I clinical trial. Results: The main organs that accumulated [{sup 99m}Tc]rBitistatin were kidneys, liver and spleen in all animal species and humans. The main organs seen on human images were the kidneys and spleen. Liver uptake was fainter, and soft-tissue background was low. [{sup 99m}Tc]rBitistatin bound to circulating platelets in blood, with a higher percentage of binding to platelets in guinea pigs and dogs compared to that in humans. Plasma protein binding was low and of little consequence in view of platelet binding. The main route of excretion was through the urine. [{sup 99m}Tc]rBitistatin did not affect platelet counts in humans or dogs. Conclusions: [{sup 99m}Tc]rBitistatin, when administered at low doses for imaging, has no adverse effects on platelets and has the qualitative biodistribution predicted by animal studies. [{sup 99m}Tc]rBitistatin was found to bind to circulating platelets in humans, suggesting that it will be able to bind

  18. A comparative study of myosin and its subunits in adult and neonatal-rat hearts and in rat heart cells from young and old cultures.

    OpenAIRE

    Ghanbari, H A; McCarl, R L

    1980-01-01

    A possible explanation for the decrease in myosin Ca2+-dependent ATPase activity as rat heart cells age in culture is presented. The subunit structure and enzyme kinetics of myosin from adult and neonatal rat hearts and from rat heart cells of young and old cultures are compared. These studies indicate that the loss in Ca-ATPase activity of myosin from older cultures was an intrinsic property of the myosin itself. Myofibrillar fractions from the indicated four sources showed no qualitative or...

  19. Evidence for alpha-MSH binding sites on human scalp hair follicles: preliminary results

    NARCIS (Netherlands)

    Nanninga, P. B.; Ghanem, G. E.; Lejeune, F. J.; Bos, J. D.; Westerhof, W.

    1991-01-01

    Alpha-MSH, considered an important pigmentation hormone, binds to melanocytes and is thought to stimulate melanogenesis through a cyclic-AMP-dependent mechanism. The binding of alpha-MSH to follicular melanocytes has been investigated in human hair of different colors, ranging from black to blond

  20. Skeletal muscle myosin heavy chain isoform content in relation to gonadal hormones and anabolic-catabolic balance in trained and untrained men.

    Science.gov (United States)

    Grandys, Marcin; Majerczak, Joanna; Karasinski, Janusz; Kulpa, Jan; Zoladz, Jerzy A

    2012-12-01

    Gonadal hormones and anabolic-catabolic hormone balance have potent influence on skeletal muscle tissue, but little is known about their action with regard to myosin heavy chain (MHC) transformation in humans. We investigated the relationship between skeletal muscle MHC isoform content in the vastus lateralis muscle and basal testosterone (T) concentration in 3 groups of subjects: endurance trained (E), sprint/strength trained (S), and untrained (U) young men. We have also determined basal sex hormone-binding globulin and cortisol (C) concentrations in untrained subjects to examine the relationship between MHC composition and the anabolic-catabolic hormone balance. Moreover, basal free testosterone (fT) and bioavailable testosterone (bio-T) concentrations were calculated for this subgroup. Despite significant differences in MHC isoform content (69.4 ± 2.39%, 61.4 ± 8.04%, and 37.5 ± 13.80% of MHC-2 for groups S, U, and E, respectively, Kruskal-Wallis: H = 18.58, p 0.5). We have also found that in the U group, type 2 MHC in the vastus lateralis muscle is positively correlated with basal fT:C ratio (r = 0.63, p = 0.01). It is concluded that the differences in the training history and training specificity can be distinguished with regard to the MHC composition but not with regard to the basal T concentration. Simultaneously, it has been shown that MHC isoform content in human vastus lateralis muscle may be related to basal anabolic-catabolic hormone balance, and this hypothesis needs further investigation.

  1. The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor

    Directory of Open Access Journals (Sweden)

    Kratochwil Nicole A

    2007-10-01

    Full Text Available Abstract Background Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone. Results By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore. Conclusion From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor

  2. Visualization of specific binding sites of benzodiazepine in human brain

    International Nuclear Information System (INIS)

    Shinotoh, H.; Yamasaki, T.; Inoue, O.; Itoh, T.; Suzuki, K.; Hashimoto, K.; Tateno, Y.; Ikehira, H.

    1986-01-01

    Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of [11C]Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that [11C] Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans

  3. Pattern of human chorionic gonadotropin binding in the polycystic ovary

    International Nuclear Information System (INIS)

    Brawer, J.; Richard, M.; Farookhi, R.

    1989-01-01

    The histologic evolution of polycystic ovaries in the estradiol valerate-treated rat coincides with the development of a unique plasma pattern of luteinizing hormone. To assess the role of luteinizing hormone in polycystic ovaries, it is necessary to evaluate the luteinizing hormone sensitivity of the specific tissues in the polycystic ovary. Therefore, we examined the pattern of luteinizing hormone binding sites in polycystic ovaries. Rats at 4 or 8 weeks after estradiol valerate treatment each received an intrajugular injection of iodine 125-labeled human chorionic gonadotropin. Some rats also received a 1000-fold excess of unlabeled human chorionic gonadotropin in the same injection. Ovaries were prepared for autoradiography. Dense accumulations of grains occurred over the theca of normal and atretic secondary follicles in all ovaries and over clusters of secondary interstitial cells. The iodine label was variable over the typically hypertrophied theca of precystic follicles. The theca of definitive cysts showed little or no label. These results indicate that cyst formation coincides with the loss of luteinizing hormone/human chorionic gonadotropin binding to the affected follicles

  4. Pattern of human chorionic gonadotropin binding in the polycystic ovary

    Energy Technology Data Exchange (ETDEWEB)

    Brawer, J.; Richard, M.; Farookhi, R. (McGill Univ., Montreal, Quebec (Canada))

    1989-08-01

    The histologic evolution of polycystic ovaries in the estradiol valerate-treated rat coincides with the development of a unique plasma pattern of luteinizing hormone. To assess the role of luteinizing hormone in polycystic ovaries, it is necessary to evaluate the luteinizing hormone sensitivity of the specific tissues in the polycystic ovary. Therefore, we examined the pattern of luteinizing hormone binding sites in polycystic ovaries. Rats at 4 or 8 weeks after estradiol valerate treatment each received an intrajugular injection of iodine 125-labeled human chorionic gonadotropin. Some rats also received a 1000-fold excess of unlabeled human chorionic gonadotropin in the same injection. Ovaries were prepared for autoradiography. Dense accumulations of grains occurred over the theca of normal and atretic secondary follicles in all ovaries and over clusters of secondary interstitial cells. The iodine label was variable over the typically hypertrophied theca of precystic follicles. The theca of definitive cysts showed little or no label. These results indicate that cyst formation coincides with the loss of luteinizing hormone/human chorionic gonadotropin binding to the affected follicles.

  5. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  6. The fungal myosin I is essential for Fusarium toxisome formation

    Science.gov (United States)

    The mycotoxin deoxynivalenol (DON) is the most frequently detected secondary metabolite produced by Fusarium graminearum and other Fusarium spp. To date, relatively few studies have addressed how mycotoxin biosynthesis occurs in fungal cells. Here we found that myosin I governs translation of DON bi...

  7. Demonstration of specific binding sites for 3H-RRR-alpha-tocopherol on human erythrocytes

    International Nuclear Information System (INIS)

    Kitabchi, A.E.; Wimalasena, J.

    1982-01-01

    Previous work from our laboratory demonstrated specific binding sites for 3 H-RRR-alpha-tocopherol ( 3 H-d alpha T) in membranes of rat adrenal cells. As tocopherol deficiency is associated with increased susceptibility of red blood cells to hemolysis, we investigated tocopherol binding sites in human RBCs. Erythrocytes were found to have specific binding sites for 3 H-d alpha T that exhibited saturability and time and cell-concentration dependence as well as reversibility of binding. Kinetic studies of binding demonstrated two binding sites--one with high affinity (Ka of 2.6 x 10(7) M-1), low capacity (7,600 sites per cell) and the other with low affinity (1.2 x 10(6) M-1), high capacity (150,000 sites per cell). In order to localize the binding sites further, RBCs were fractionated and greater than 90% of the tocopherol binding was located in the membranes. Similar to the findings in intact RBCs, the membranes exhibited two binding sites with a respective Ka of 3.3 x 10(7) M-1 and 1.5 x 10(6) M-1. Specificity data for binding demonstrated 10% binding for RRR-gamma-tocopherol, but not other tocopherol analog exhibited competition for 3 H-d alpha T binding sites. Instability data suggested a protein nature for these binding sites. Preliminary studies on Triton X-100 solubilized fractions resolved the binding sites to a major component with an Mr of 65,000 and a minor component with an Mr of 125,000. We conclude that human erythrocyte membranes contain specific binding sites for RRR-alpha-tocopherol. These sites may be of physiologic significance in the function of tocopherol on the red blood cell membrane

  8. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    International Nuclear Information System (INIS)

    MacDonald, P.N.; Ong, D.E.; Bok, D.

    1990-01-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur

  9. Synthesis of total protein (TP) and myosin heavy chain (HC) isozymes in pressure overloaded rabbit hearts

    International Nuclear Information System (INIS)

    Nagai, R.; Martin, B.J.; Pritzl, N.; Zak, R.; Low, R.B.; Stirewalt, W.S.; Alpert, N.R.; Litten, R.Z.

    1986-01-01

    Pulmonary artery banding (PO) leads to a rapid increase in right ventricular (RV) weight as well as a shift toward β myosin isozyme. They determined: (1) the contributions of changes in the capacity (RNA content) and efficiency of total protein synthesis to the increase in RV weight; and (2) the relative contributions of translational and pretranslational mechanisms to the shift in myosin HC isotypes. The rates of synthesis in vivo of TP, α- and β-HC were measured by a constant infusion technique using 3 H-leucine. TP synthesis was 7 +/- 2(SD) mg/day in control (RV:367 +/- 70 mg) and was increased by 2.6 fold at day 2 and 2.9 fold at day 4 following PO (p < 0.01). RV RNA content was increased by 83% at day 2 and 103% at day 4 PO (p < 0.05). The efficiency of synthesis (rate/RNA) was also significantly higher at these time points (1.4- and 1.3-fold). β-HC synthesis was 0.6 +/- 0.2 mg/day in control and increased by 2.6 fold at day 2 and 3.5 fold at day 4 following PO. In contrast, the rate of synthesis of α-HC was unchanged. The relative rates of β-HC to total HC synthesis was correlated linearly with the relative levels of β-myosin mRNA as measured by S1 nuclease mapping. They conclude that increases in the proportion of β-HC myosin following PO is due to increases in the relative amount of β-myosin mRNA and therefore involves modulation of a pretranslational mechanism

  10. Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level

    Science.gov (United States)

    SERRANO, A. L.; PÉREZ, MARGARITA; LUCÍA, A.; CHICHARRO, J. L.; QUIROZ-ROTHE, E.; RIVERO, J. L. L.

    2001-01-01

    The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus lateralis muscle biopsies of 15 young men (with an average age of 22 y) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC β-slow, MHC-IIA and MHC-IIX. The characterisation of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories according to the MHC isoform they express at the protein level, types I, I+IIA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were clearly underestimated by mATPase histochemistry. For a very high percentage of fibres there was a precise correspondence between the MHC protein isoforms and mRNA transcripts. The integrated methods used demonstrate a high degree of precision of the immunohistochemical procedure used for the identification and quantification of human skeletal muscle fibre types. The monoclonal antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibres. This protocol offers new prospects for muscle fibre classification in human experimental studies. PMID:11554510

  11. Myosin XI-Dependent Formation of Tubular Structures from Endoplasmic Reticulum Isolated from Tobacco Cultured BY-2 Cells1[W][OA

    Science.gov (United States)

    Yokota, Etsuo; Ueda, Haruko; Hashimoto, Kohsuke; Orii, Hidefumi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Shimmen, Teruo

    2011-01-01

    The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER. PMID:21427277

  12. Alterations in rat cardiac myosin isozymes induced by whole-body irradiation are prevented by 3,5,3'-L-triiodothyronine

    Energy Technology Data Exchange (ETDEWEB)

    Litten, R.Z.; Fein, H.G.; Gainey, G.T.; Walden, T.L.; Smallridge, R.C. (Armed Forces Radiobiology Research Institute, Bethesda, MD (USA))

    1990-01-01

    Changes in cardiac myosin isozymes and serum thyroid hormone levels were investigated in rats following 10 Gy whole-body gamma irradiation. The percent beta-myosin heavy chain increased from 21.3 {plus minus} 1.8 to 28.1 {plus minus} 6.8 (NS) at 3-day postirradiation, 37.7 {plus minus} 1.9 (P less than .001) at 6-day postirradiation, and 43.8 {plus minus} 3.3 (P less than .001) at 9-day postirradiation. Along with the change in myosin isozymes was a significant 53% decrease (P less than .001) in the serum thyroxine (T4) level by day 3 postirradiation, remaining depressed through day 9 postirradiation. The serum 3,5,3'-triiodothyronine (T3) level, however, was normal until day 9, when significant depression was also observed. In contrast, the thyroid-stimulating hormone (TSH) level was significantly increased by fourfold at day 3, returning to near normal values by day 9 postirradiation. Daily injections of physiological doses of T3 (0.3 microgram/100 g body weight) prevented the change in the myosin isozymes following whole-body irradiation. Daily pharmacological injections of T3 (3.0 micrograms/100 g body weight) to the irradiated rats produced a further decrease in the percent beta-myosin heavy chain (below control values) indicating tissue hyperthyroidism. Thus, this study suggests that the change in myosin isozymes following whole-body irradiation is caused by an alteration in thyroid hormone activity.

  13. Stable and dynamic microtubules coordinately shape the myosin activation zone during cytokinetic furrow formation

    Science.gov (United States)

    Foe, Victoria E.; von Dassow, George

    2008-01-01

    The cytokinetic furrow arises from spatial and temporal regulation of cortical contractility. To test the role microtubules play in furrow specification, we studied myosin II activation in echinoderm zygotes by assessing serine19-phosphorylated regulatory light chain (pRLC) localization after precisely timed drug treatments. Cortical pRLC was globally depressed before cytokinesis, then elevated only at the equator. We implicated cell cycle biochemistry (not microtubules) in pRLC depression, and differential microtubule stability in localizing the subsequent myosin activation. With no microtubules, pRLC accumulation occurred globally instead of equatorially, and loss of just dynamic microtubules increased equatorial pRLC recruitment. Nocodazole treatment revealed a population of stable astral microtubules that formed during anaphase; among these, those aimed toward the equator grew longer, and their tips coincided with cortical pRLC accumulation. Shrinking the mitotic apparatus with colchicine revealed pRLC suppression near dynamic microtubule arrays. We conclude that opposite effects of stable versus dynamic microtubules focuses myosin activation to the cell equator during cytokinesis. PMID:18955555

  14. Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Lantz, M.S.; Allen, R.D.; Vail, T.A.; Switalski, L.M.; Hook, M. (Univ. of Alabama at Birmingham (USA))

    1991-01-01

    Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains. The authors now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37{degree}C. A functional fibrinogen-binding component (M{sub r}, 150 000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with {sup 125}I-fibrinogen. Fibrinogen degradation did not occur at 4{degree}C but did occur at 22 and 37{degree}C. When bacteria and iodinated fibrinogen were incubated at 37{degree}C, two major fibrinogen fragments (M{sub r}, 97 000 and 50 000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (M{sub r}, 120 000 and 150 000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the M{sub r}-120 000 and -150 000 proteases was enhanced by reducing agents, completely inhibited by N-{alpha}-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate.

  15. Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen

    International Nuclear Information System (INIS)

    Lantz, M.S.; Allen, R.D.; Vail, T.A.; Switalski, L.M.; Hook, M.

    1991-01-01

    Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains. The authors now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37 degree C. A functional fibrinogen-binding component (M r , 150 000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with 125 I-fibrinogen. Fibrinogen degradation did not occur at 4 degree C but did occur at 22 and 37 degree C. When bacteria and iodinated fibrinogen were incubated at 37 degree C, two major fibrinogen fragments (M r , 97 000 and 50 000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (M r , 120 000 and 150 000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the M r -120 000 and -150 000 proteases was enhanced by reducing agents, completely inhibited by N-α-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate

  16. Human myometrial adrenergic receptors during pregnancy: identification of the alpha-adrenergic receptor by [3H] dihydroergocryptine binding

    International Nuclear Information System (INIS)

    Jacobs, M.M.; Hayashida, D.; Roberts, J.M.

    1985-01-01

    The radioactive alpha-adrenergic antagonist [ 3 H] dihydroergocryptine binds to particulate preparations of term pregnant human myometrium in a manner compatible with binding to the alpha-adrenergic receptor (alpha-receptor). [ 3 H] Dihydroergocryptine binds with high affinity (KD = 2 nmol/L and low capacity (receptor concentration = 100 fmol/mg of protein). Adrenergic agonists compete for [ 3 H] dihydroergocryptine binding sites stereo-selectively ([-]-norepinephrine is 100 times as potent as [+]-norepinephrine) and in a manner compatible with alpha-adrenergic potencies (epinephrine approximately equal to norepinephrine much greater than isoproterenol). Studies in which prazosin, an alpha 1-antagonist, and yohimbine, and alpha 2-antagonist, competed for [ 3 H] dihydroergocryptine binding sites in human myometrium indicated that approximately 70% are alpha 2-receptors and that 30% are alpha 1-receptors. [ 3 H] dihydroergocryptine binding to human myometrial membrane particulate provides an important tool with which to study the molecular mechanisms of uterine alpha-adrenergic response

  17. Human mast cell neutral proteases generate modified LDL particles with increased proteoglycan binding.

    Science.gov (United States)

    Maaninka, Katariina; Nguyen, Su Duy; Mäyränpää, Mikko I; Plihtari, Riia; Rajamäki, Kristiina; Lindsberg, Perttu J; Kovanen, Petri T; Öörni, Katariina

    2018-04-13

    Subendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. Mature human MCs were differentiated from human peripheral blood-derived CD34 + progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. Activated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. The present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. CCL2 binding is CCR2 independent in primary adult human astrocytes.

    Science.gov (United States)

    Fouillet, A; Mawson, J; Suliman, O; Sharrack, B; Romero, I A; Woodroofe, M N

    2012-02-09

    Chemokines are low relative molecular mass proteins, which have chemoattractant actions on many cell types. The chemokine, CCL2, has been shown to play a major role in the recruitment of monocytes in central nervous system (CNS) lesions in multiple sclerosis (MS). Since resident astrocytes constitute a major source of chemokine synthesis including CCL2, we were interested to assess the regulation of CCL2 by astrocytes. We showed that CCL2 bound to the cell surface of astrocytes and binding was not modulated by inflammatory conditions. However, CCR2 protein was not detected nor was activation of the classical CCR2 downstream signaling pathways. Recent studies have shown that non-signaling decoy chemokine receptors bind and modulate the expression of chemokines at site of inflammation. Here, we show that the D6 chemokine decoy receptor is constitutively expressed by primary human adult astrocytes at both mRNA and protein level. In addition, CCL3, which binds to D6, but not CCL19, which does not bind to D6, displaced CCL2 binding to astrocytes; indicating that CCL2 may bind to this cell type via the D6 receptor. Our results suggest that CCL2 binding to primary adult human astrocytes is CCR2-independent and is likely to be mediated via the D6 decoy chemokine receptor. Therefore we propose that astrocytes are implicated in both the establishment of chemokine gradients for the migration of leukocytes into and within the CNS and in the regulation of CCL2 levels at inflammatory sites in the CNS. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX.

    Science.gov (United States)

    Linkuvienė, Vaida; Matulienė, Jurgita; Juozapaitienė, Vaida; Michailovienė, Vilma; Jachno, Jelena; Matulis, Daumantas

    2016-04-01

    Human carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding. The observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry. The pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was -24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM. The intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship. It is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Human Islet Amyloid Polypeptide Fibril Binding to Catalase: A Transmission Electron Microscopy and Microplate Study

    Directory of Open Access Journals (Sweden)

    Nathaniel G. N. Milton

    2010-01-01

    Full Text Available The diabetes-associated human islet amyloid polypeptide (IAPP is a 37-amino-acid peptide that forms fibrils in vitro and in vivo. Human IAPP fibrils are toxic in a similar manner to Alzheimer's amyloid-β (Aβ and prion protein (PrP fibrils. Previous studies have shown that catalase binds to Aβ fibrils and appears to recognize a region containing the Gly-Ala-Ile-Ile sequence that is similar to the Gly-Ala-Ile-Leu sequence found in human IAPP residues 24-27. This study presents a transmission electron microscopy (TEM—based analysis of fibril formation and the binding of human erythrocyte catalase to IAPP fibrils. The results show that human IAPP 1-37, 8-37, and 20-29 peptides form fibrils with diverse and polymorphic structures. All three forms of IAPP bound catalase, and complexes of IAPP 1-37 or 8-37 with catalase were identified by immunoassay. The binding of biotinylated IAPP to catalase was high affinity with a KD of 0.77nM, and could be inhibited by either human or rat IAPP 1-37 and 8-37 forms. Fibrils formed by the PrP 118-135 peptide with a Gly-Ala-Val-Val sequence also bound catalase. These results suggest that catalase recognizes a Gly-Ala-Ile-Leu—like sequence in amyloid fibril-forming peptides. For IAPP 1-37 and 8-37, the catalase binding was primarily directed towards fibrillar rather than ribbon-like structures, suggesting differences in the accessibility of the human IAPP 24-27 Gly-Ala-Ile-Leu region. This suggests that catalase may be able to discriminate between different structural forms of IAPP fibrils. The ability of catalase to bind IAPP, Aβ, and PrP fibrils demonstrates the presence of similar accessible structural motifs that may be targets for antiamyloid therapeutic development.

  1. Non-immune binding of human IgG to M-related proteins confers resistance to phagocytosis of group A streptococci in blood.

    Directory of Open Access Journals (Sweden)

    Harry S Courtney

    Full Text Available The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp are group A streptococcal (GAS receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG₁>IgG₄>IgG₂>IgG₃. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.

  2. Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose

    DEFF Research Database (Denmark)

    Kirkeby, S

    2016-01-01

    FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini...... to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human...

  3. Botulinum Toxin Type A Inhibits α-Smooth Muscle Actin and Myosin II Expression in Fibroblasts Derived From Scar Contracture.

    Science.gov (United States)

    Chen, Minliang; Yan, Tongtong; Ma, Kui; Lai, Linying; Liu, Chang; Liang, Liming; Fu, Xiaobing

    2016-09-01

    Scar contracture (SC) is one of the most common complications resulting from major burn injuries. Numerous treatments are currently available but they do not always yield excellent therapeutic results. Recent reports suggest that botulinum toxin type A (BTXA) is effective at reducing SC clinically, but the molecular mechanism for this action is unknown. α-Smooth muscle actin (α-SMA) and myosin II are the main components of stress fibers, which are the contractile structures of fibroblasts. The effects of BTXA on α-SMA and myosin II in SC are still unknown. This study aimed to explore the effect of BTXA on α-SMA and myosin II expression in fibroblasts derived from SC and to elucidate its actual mechanism further. Fibroblasts were isolated from tissue specimens of SC. Fibroblasts were cultured in Dulbecco modified Eagle medium with different concentrations of BTXA and their proliferation was analyzed through the tetrazolium-based colorimetric method at 1, 4, and 7 days. Proteins of α-SMA and myosin II were checked using Western blot in fibroblasts treated with different concentrations of BTXA at 1, 4, and 7 days. Fibroblasts without BTXA treatment had a higher proliferation than that in other groups, which indicated that the proliferation of fibroblasts was significantly inhibited by BTXA (P < 0.05). Proteins of α-SMA and myosin II between fibroblasts with BTXA and fibroblasts without BTXA are statistically significant (P < 0.05). These results suggest that BTXA effectively inhibited the growth of fibroblasts derived from SC and reduced the expression of α-SMA and myosin II, which provided theoretical support for the application of BTXA to control SC.

  4. Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

    NARCIS (Netherlands)

    Wilczak, N; Kuhl, N; Chesik, D; Geerts, A; Luiten, P; De Keyser, J

    The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R-3 -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were

  5. A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

    Science.gov (United States)

    Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

    2015-02-01

    Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. © 2014 Society for Laboratory Automation and Screening.

  6. 17β-Estradiol-induced interaction of estrogen receptor α and human atrial essential myosin light chain modulates cardiac contractile function.

    Science.gov (United States)

    Duft, Karolin; Schanz, Miriam; Pham, Hang; Abdelwahab, Ahmed; Schriever, Cindy; Kararigas, Georgios; Dworatzek, Elke; Davidson, Mercy M; Regitz-Zagrosek, Vera; Morano, Ingo; Mahmoodzadeh, Shokoufeh

    2017-01-01

    Chronic increased workload of the human heart causes ventricular hypertrophy, re-expression of the atrial essential myosin light chain (hALC-1), and improved contractile function. Although hALC-1 is an important positive inotropic regulator of the human heart, little is known about its regulation. Therefore, we investigated the role of the sex hormone 17β-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function. We showed that E2 attenuated hALC-1 expression in human atrial tissues of both sexes and in human ventricular AC16 cells. E2 induced the nuclear translocation of estrogen receptor alpha (ERα) and hALC-1 in AC16 cells, where they cooperatively regulate the transcriptional activity of hALC-1 gene promoter. E2-activated ERα required the estrogen response element (ERE) motif within the hALC-1 gene promoter to reduce its transcriptional activity (vehicle: 15.55 ± 4.80 vs. E2: 6.51 ± 3.69; ~2 fold). This inhibitory effect was potentiated in the presence of hALC-1 (vehicle: 11.13 ± 3.66 vs. E2: 2.18 ± 1.10; ~5 fold), and thus, hALC-1 acts as a co-repressor of ERα-mediated transcription. Yeast two-hybrid screening of a human heart cDNA library revealed that ERα interacts physically with hALC-1 in the presence of E2. This interaction was confirmed by Co-Immunoprecipitation and immunofluorescence in human atrium. As a further novel effect, we showed that chronic E2-treatment of adult mouse cardiomyocytes overexpressing hALC-1 resulted in reduced cell-shortening amplitude and twitching kinetics of these cells independent of Ca 2+ activation levels. Together, our data showed that the expression of hALC-1 gene is, at least partly, regulated by E2/ERα, while hALC-1 acts as a co-repressor. The inotropic effect of hALC-1 overexpression in cardiomyocytes can be significantly repressed by E2.

  7. Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Mercurio, A M; Chung, S Y

    1990-01-01

    Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from...... was then isolated and used to elicit a rabbit antiserum. In immunostaining, both antisera reacted with the nuclei of cultured tumor cells. In tissue sections of human carcinoma, nuclear immunoreactivity was observed in the tumor cells in 40 of 42 cases examined. Adjacent normal epithelial tissue obtained from......, the presence of the homeobox transcript in human carcinoma was documented by in situ hybridization and RNase protection mapping. These results demonstrate that human cancer is associated with the expression of homeobox proteins. Such homeobox proteins, as well as other regulatory proteins, could be involved...

  8. Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport.

    Science.gov (United States)

    Stapleton, Nigel M; Armstrong-Fisher, Sylvia S; Andersen, Jan Terje; van der Schoot, C Ellen; Porter, Charlene; Page, Kenneth R; Falconer, Donald; de Haas, Masja; Williamson, Lorna M; Clark, Michael R; Vidarsson, Gestur; Armour, Kathryn L

    2018-03-01

    We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half-life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto-maternal alloimmune disorders. Here, we show that G1Δnab mutants retain pH-dependent binding to human FcRn but that the amino acid alterations reduce the affinity of the IgG1:FcRn interaction by 2.0-fold and 1.6-fold for the two antibodies investigated. The transport of the modified G1Δnab mutants across monolayers of human cell lines expressing FcRn was approximately 75% of the wild-type, except that no difference was observed with human umbilical vein endothelial cells. G1Δnab mutation also reduced transport in an ex vivo placenta model. In conclusion, we demonstrate that, although the G1Δnab mutations are away from the FcRn-binding site, they have long-distance effects, modulating FcRn binding and transcellular transport. Our findings have implications for the design of therapeutic human IgG with tailored effector functions. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis...

  10. Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers.

    Science.gov (United States)

    Arata, T

    1990-07-20

    Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers. e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 degrees relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spin-labeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 degrees and 66 degrees, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5'-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.

  11. Human platelet ( sup 125 I)R-DOI binding sites. Characterization by in vitro autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Himeno, A.; Saavedra, J.M. (National Institute of Mental Health, Bethesda, MD (USA))

    1990-02-01

    We quantified binding sites for 2,5-dimethoxy-4-iodo-phenylisopropylamine (DOI), a 5-HT2 agonist and hallucinogen, in human platelets. We incubated sections from human platelet pellets with ({sup 125}I)R-DOI with or without 1 mumol/L ketanserin, followed by autoradiography and computerized microdensitometry. We corrected the values of binding density by the protein content of each section with a densitometric protein assay. The present method revealed a single class of high affinity binding sites for ({sup 125}I)R-DOI, with a Kd of 6.4 +/- 0.7 nmol/L and a Bmax of 100 +/- 10 fmol/mg protein. Kd and Bmax for ({sup 125}I)R-DOI determined by the classical membrane binding assay, were 2.7 +/- 0.4 nmol/L and 100 +/- 10 fmol/mg protein, respectively. The present method is precise, very sensitive, and allows the characterization of ({sup 125}I)R-DOI binding in sections obtained from as little as 3 ml of blood. Standardization is possible after correction by the protein content of each individual section.

  12. Structural and Histone Binding Ability Characterizations of Human PWWP Domains

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong (Toronto); (Penn)

    2013-09-25

    The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

  13. Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible

    International Nuclear Information System (INIS)

    Innis, W.S.; McCormick, D.B.; Merrill, A.H. Jr.

    1985-01-01

    Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient

  14. Autoradiographic localization of calcitonin gene-related peptide (CGRP) binding sites in human and guinea pig lung

    International Nuclear Information System (INIS)

    Mak, J.C.; Barnes, P.J.

    1988-01-01

    125 I-Human calcitonin gene-related peptide (hCGRP) binding sites were localized in human and guinea pig lungs by an autoradiographic method. Scatchard analysis of saturation experiments from slide-mounted sections of guinea pig lung displayed specific 125 I-hCGRP binding sites with a dissociation constant (Kd) of 0.72 +/- 0.05 nM (mean +/- S.E.M., n = 3) and a maximal number of binding sites (Bmax) of 133.4 +/- 5.6 fmol/mg protein. In both human and guinea pig lung, autoradiography revealed that CGRP binding sites were widely distributed, with particularly dense labeling over bronchial and pulmonary blood vessels of all sizes and alveolar walls. Airway smooth muscle and epithelium of large airways was sparsely labeled but no labeling was found over submucosal glands. This localization corresponds well to the reported pattern of CGRP-like immunoreactive innervation. The findings of localization of CGRP binding sites on bronchial and pulmonary blood vessels indicate that CGRP may be important in the regulation of airway and pulmonary blood flow

  15. Probing the binding of vitexin to human serum albumin by multispectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Guowen, E-mail: gwzhang@ncu.edu.c [State Key Laboratory of Food Science and Technology, Nanchang University, 235, Nanjing East Road, Nanchang 330047, Jiangxi (China); Zhao Nan; Wang Lin [State Key Laboratory of Food Science and Technology, Nanchang University, 235, Nanjing East Road, Nanchang 330047, Jiangxi (China)

    2011-05-15

    The interaction between vitexin and human serum albumin (HSA) has been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy under simulated physiological conditions. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of vitexin to HSA. The binding constants (K{sub a}) between vitexin and HSA were obtained according to the modified Stern-Volmer equation. The thermodynamic parameters, enthalpy change ({Delta}H) and entropy change ({Delta}S) were calculated to be -57.29 kJ mol{sup -1} and -99.01 J mol{sup -1} K{sup -1} via the van't Hoff equation, which indicated that the interaction of vitexin with HSA was driven mainly by hydrogen bond and van der Waals forces. Fluorescence anisotropy data showed that warfarin and vitexin shared a common binding site I corresponding to the subdomain IIA of HSA. The binding distance (r) between the donor (HSA) and the acceptor (vitexin) was 4.16 nm based on the Foerster theory of non-radioactive energy transfer. In addition, the results of synchronous fluorescence, CD and FT-IR spectra demonstrated that the microenvironment and the secondary structure of HSA were changed in the presence of vitexin. - Research highlights: We investigate the binding mechanism of vitexin to human serum albumin (HSA) by different multi-spectroscopic techniques under simulated physiological conditions. Vitexin can strongly quench the fluorescence of HSA through a static quenching mechanism. The interaction of vitexin with HSA is driven mainly by hydrogen bond and van der Waals forces. The binding distance between HSA and vitexin is 4.16 nm, and vitexin is mainly located in the region of site I (subdomain IIA). The binding of vitexin to HSA can induce conformational changes of HSA.

  16. Cyclophilin B binding to platelets supports calcium-dependent adhesion to collagen.

    Science.gov (United States)

    Allain, F; Durieux, S; Denys, A; Carpentier, M; Spik, G

    1999-08-01

    We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 +/- 3 nmol/L and the number of sites estimated at 960 +/- 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca(2+) and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca(2+) channel.

  17. Analysis of positional isotope exchange in ATP by cleavage of the βP-OγP bond. Demonstration of negligible positional isotope exchange by myosin

    International Nuclear Information System (INIS)

    Dale, M.P.; Hackney, D.D.

    1987-01-01

    A method for analysis of positional isotope exchange (PIX) during ATP ↔ HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the βP-OγP bond. This cleavage yields P/sub i/ derived from the γ-phosphoryl of ATP that contains all four of the γ oxygens. Both PIX between the β, γ-bridge and β-nonbridge positions and washout of the γ-nonbridge oxygens can be simultaneously followed by using ATP labeled with 17 O at the β-nonbridge positions and 18 O at the β,γ-bridge and γ-nonbridge positions. Application of this method to ATP ↔ HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of γ-nonbridge oxygens in the virtual absence of PIX. At 25 0 C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0.001 s -1 , which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the β-oxygens of bound ADP of 1 in 10,000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does not undergo rapid washout. With myosin and stoichiometric ATP at 0 0 C, this fraction correspond to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with subfragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanation for the PIX observed by Geeves et al. with subfragment 1

  18. Antigen-binding radioimmunoassays for human IgG antibodies to bovine ν-lactoglobulin

    International Nuclear Information System (INIS)

    Turner, M.W.; Paganelli, R.; Levinsky, R.J.; Williams, A.

    1983-01-01

    A double antibody antigen-binding assay for the detection of human IgG antibodies to the bovine milk allergen ν-lactoglobulin is described. The levels of such antibodies in patients with established cows' milk protein intolerance were significantly higher than the levels observed in a healthy control group (P<0.01). The assay showed excellent correlation with a solid phase antigen binding assay (rsub(s) = 0.8, P<0.001). (Auth.)

  19. Standardization for cortisol determination in human blood by competitive protein-binding

    International Nuclear Information System (INIS)

    Okada, H.

    1978-01-01

    Standardization for determination of cortisol from human plasma (17-hydroxycorticosteroids) using competitive protein-binding method is presented. Activated carbon coated with dextrans is used for separation of the hormone-protein complexe and hormone labelled free [pt

  20. Analysis of the binding interaction in uric acid - Human hemoglobin system by spectroscopic techniques

    Science.gov (United States)

    Makarska-Bialokoz, Magdalena

    2017-05-01

    The binding interaction between human hemoglobin and uric acid has been studied for the first time, by UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence techniques. Characteristic effects observed for human hemoglobin intrinsic fluorescence during interaction with uric acid at neutral pH point at the formation of stacking non-covalent and non-fluorescent complexes. All the calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants, as well as Förster resonance energy transfer parameters confirm the existence of static quenching. The results of synchronous fluorescence measurements indicate that the fluorescence quenching of human hemoglobin originates both from Trp and Tyr residues and that the addition of uric acid could significantly hinder the physiological functions of human hemoglobin.

  1. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): Identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42

    International Nuclear Information System (INIS)

    Shinjo, K.; Koland, J.G.; Hart, M.J.; Narasimhan, V.; Cerione, R.A.; Johnson, D.I.; Evans, T.

    1990-01-01

    The authors have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated G p (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human G p protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins and the rac proteins. The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell division-cycle protein CDC42. The human placental gene, which they designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein

  2. Structural basis for antagonism of human interleukin 18 by poxvirus interleukin 18-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Krumm, Brian; Meng, Xiangzhi; Li, Yongchao; Xiang, Yan; Deng, Junpeng (Texas-HSC); (OKLU)

    2009-07-10

    Human interleukin-18 (hIL-18) is a cytokine that plays an important role in inflammation and host defense against microbes. Its activity is regulated in vivo by a naturally occurring antagonist, the human IL-18-binding protein (IL-18BP). Functional homologs of human IL-18BP are encoded by all orthopoxviruses, including variola virus, the causative agent of smallpox. They contribute to virulence by suppressing IL-18-mediated immune responses. Here, we describe the 2.0-{angstrom} resolution crystal structure of an orthopoxvirus IL-18BP, ectromelia virus IL-18BP (ectvIL-18BP), in complex with hIL-18. The hIL-18 structure in the complex shows significant conformational change at the binding interface compared with the structure of ligand-free hIL-18, indicating that the binding is mediated by an induced-fit mechanism. EctvIL-18BP adopts a canonical Ig fold and interacts via one edge of its {beta}-sandwich with 3 cavities on the hIL-18 surface through extensive hydrophobic and hydrogen bonding interactions. Most of the ectvIL-18BP residues that participate in these interactions are conserved in both human and viral homologs, explaining their functional equivalence despite limited sequence homology. EctvIL-18BP blocks a putative receptor-binding site on IL-18, thus preventing IL-18 from engaging its receptor. Our structure provides insights into how IL-18BPs modulate hIL-18 activity. The revealed binding interface provides the basis for rational design of inhibitors against orthopoxvirus IL-18BP (for treating orthopoxvirus infection) or hIL-18 (for treating certain inflammatory and autoimmune diseases).

  3. Characterization and immunohistochemical localization of rat salivary cobalamin-binding protein and comparison with human salivary haptocorrin

    DEFF Research Database (Denmark)

    Nexø, Ebba; Poulsen, Steen Seier

    1985-01-01

    Rat saliva contains a cobalamin-binding protein that binds cobalamin as well as cobinamide. The protein binds cobalamin with an affinity constant of 8 X 10(10) l X mol-1, and it binds cobalamin over a more narrow pH range (pH 7.5-10) than does human haptocorrin. It has a Stokes radius of 2.45 nm...

  4. Serotonin binding in vitro by releasable proteins from human blood platelets

    International Nuclear Information System (INIS)

    Heemstra, V.L.

    1983-11-01

    Among the substances released from human blood platelets are serotonin and various proteins. It was hypothesized that one of these proteins binds serotonin and that serotonin might be important to the protein's function or that the protein might be important to serotonin's function. Two platelet-specific proteins, platelet factor 4 (PF4) and β-thromboglobulin (βTG) were found to bind serotonin in vitro. Endogenous PF4 was isolated by serotonin-affinity chromatography and was identified by radioimmunoassay. Purified [ 125 I] -PF4 and native PF4 bound to and eluted from a serotonin-affinity column similarly. Ultrafiltration of the homologous protein, βTG, with [ 14 C]-serotonin demonstrated binding of about 8 moles serotonin per mole tetrameric βTG with a dissociation constant of about 4 X 10(sup-8) M. Equilibrium dialysis of PF4 with radiolabelled serotonin was attempted, but no binding constant values were obtained because serotonin apparently bound to the dialysis membrane. Since EDTA was one of the two agents that eluted PF4 from the serotonin-affinity gel, calcium binding by PF4 was investigated by equilibrium dialysis. Evidence was obtained for positively cooperative binding of calcium ions by PF4. It is concluded that PF4 and βTG bind serotonin in vitro, that they may also bind in vivo when platelets undergo release, and that the functions of serotonin, PF4 and βTG may be mediated in part by serotonin-protein associations

  5. Myosin content of single muscle fibers following short-term disuse and active recovery in young and old healthy men

    DEFF Research Database (Denmark)

    Hvid, Lars G; Brocca, Lorenza; Ørtenblad, Niels

    2017-01-01

    healthy men. Following disuse, myosin content decreased (p... young and old in both fiber types, with MHC 2a fibers demonstrating an overshooting in young (+31%, pStrong correlations were observed between myosin content and single fiber SF in both young and old, with greater slope steepness in MHC 2a vs 1 fibers indicating an enhanced intrinsic...

  6. Characterization of the minimum domain required for targeting budding yeast myosin II to the site of cell division

    Directory of Open Access Journals (Sweden)

    Tolliday Nicola J

    2006-06-01

    Full Text Available Abstract Background All eukaryotes with the exception of plants use an actomyosin ring to generate a constriction force at the site of cell division (cleavage furrow during mitosis and meiosis. The structure and filament forming abilities located in the C-terminal or tail region of one of the main components, myosin II, are important for localising the molecule to the contractile ring (CR during cytokinesis. However, it remains poorly understood how myosin II is recruited to the site of cell division and how this recruitment relates to myosin filament assembly. Significant conservation between species of the components involved in cytokinesis, including those of the CR, allows the use of easily genetically manipulated organisms, such as budding yeast (Saccharomyces cerevisiae, in the study of cytokinesis. Budding yeast has a single myosin II protein, named Myo1. Unlike most other class II myosins, the tail of Myo1 has an irregular coiled coil. In this report we use molecular genetics, biochemistry and live cell imaging to characterize the minimum localisation domain (MLD of budding yeast Myo1. Results We show that the MLD is a small region in the centre of the tail of Myo1 and that it is both necessary and sufficient for localisation of Myo1 to the yeast bud neck, the pre-determined site of cell division. Hydrodynamic measurements of the MLD, purified from bacteria or yeast, show that it is likely to exist as a trimer. We also examine the importance of a small region of low coiled coil forming probability within the MLD, which we call the hinge region. Removal of the hinge region prevents contraction of the CR. Using fluorescence recovery after photobleaching (FRAP, we show that GFP-tagged MLD is slightly more dynamic than the GFP-tagged full length molecule but less dynamic than the GFP-tagged Myo1 construct lacking the hinge region. Conclusion Our results define the intrinsic determinant for the localization of budding yeast myosin II and show

  7. Complexities in human herpesvirus-6A and -6B binding to host cells

    DEFF Research Database (Denmark)

    Pedersen, Simon Metz; Höllsberg, Per

    2006-01-01

    Human herpesvirus-6A and -6B uses the cellular receptor CD46 for fusion and infection of the host cell. The viral glycoprotein complex gH-gL from HHV-6A binds to the short consensus repeat 2 and 3 in CD46. Although all the major isoforms of CD46 bind the virus, certain isoforms may have higher...

  8. Characterization of binding of human alpha 2-macroglobulin to group G streptococci

    International Nuclear Information System (INIS)

    Chhatwal, G.S.; Mueller, H.P.; Blobel, H.

    1983-01-01

    An interaction was observed between human alpha 2-macroglobulin (alpha 2M) and streptococci belonging to group A, C, and G. Of 27 group C and 19 group G streptococcal cultures, 13 and 14, respectively, bound 125 I-labeled alpha 2M. Some group A streptococci also interacted with alpha 2M. A number of other bacterial species tested did not react with alpha 2M. The binding of 125 I-labeled alpha 2M to group G streptococci was time dependent, saturable, and could be inhibited by unlabeled alpha 2M. Inhibition experiments indicated that the streptococcal binding site for alpha 2M differed from the receptors for immunoglobulin G, fibrinogen, aggregated beta 2-microglobulin, albumin, and fibronectin. The alpha 2M binding activity was remarkably sensitive to trypsin and heat treatment indicating its protein nature. Kinetic analysis indicated a homogenous population of binding sites. The number of binding sites per bacterial cell was estimated to be approximately 20,000

  9. Glycine receptors in the human substantia nigra as defined by (3H)strychnine binding

    Energy Technology Data Exchange (ETDEWEB)

    de Montis, G; Beaumont, K; Javoy-Agid, F; Agid, Y; Constandinidis, J; Lowenthal, A; Lloyd, K G

    1982-03-01

    Specific (3H)strychnine binding was used to identify the glycine receptor macromolecular complex in human spinal cord, substantia nigra, inferior olivary nucleus, and cerebral cortex. In material from control patients a high-affinity KD (3--8 nM) was observed in the spinal cord and the substantia nigra, both the pars compacta and the pars reticulata. This is very similar to the values observed in the rat and bovine spinal cord (8 and 3 nM, respectively) and rat substantia nigra (12 nM). In the human brain the distribution of (3H)strychnine binding (at 10 nM) was: spinal cord . substantia nigra, pars compacta greater than substantia nigra, pars reticulata . inferior olivary nucleus greater than cerebral cortex. The binding capacity (Bmax) of the rat brain (substantia nigra or spinal cord) was approximately 10-fold that of the human brain. (3H)Strychnine binding was significantly decreased in the substantia nigra from Parkinson's disease patients, both in the pars compacta (67% of control) and the pars reticulata (50% of control), but not in the inferior olivary nucleus. The results were reproduced in preliminary experiment in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. In the substantia nigra from patients who died with Huntington's disease, (3H)strychnine binding tended to be high (150% of control, NS) in both the pars compacta and the reticulata. (3H)Strychnine binding was unaltered in the substantia nigra of patients with senile dementia. Together with previous neurophysiological and neuropharmacological findings, those results support the hypothesis of glycine receptors occurring on dopamine cell bodies and/or dendrites in the substantia nigra.

  10. Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate

    International Nuclear Information System (INIS)

    Mahmood, R.

    1985-01-01

    The photoaffinity analogue 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz 2 ATP) contains the photoreactive benzophenone group esterified at the 2' or 3' hydroxyl groups of ribose. MgBz 2 ADP has a single binding site on skeletal myosin chymotryptic subfragment-one (SF 1 ) with a binding constant of 3.2 x 10 5 M -1 . Bz 2 ATP is also a substrate for the ATPase activity of SF 1 in the presence of different cations. The irradiation of SF 1 with [ 3 H]Bz 2 ATP photoinactivates the ATPase activity with concomitant incorporation of the analogue into the enzyme. Polyacrylamide gel electrophoresis of photolabeled SF 1 after milk trypsin digestion shows that all three tryptic peptides, 25 K, 50K, and 20 K, and both light chains are labeled. The presence of ATP during irradiation reduces labeling of the 50 K peptide only indicating that the other peptides are non-specifically labeled. To reduce the non-specific labeling [ 3 H]Bz 2 ATP is trapped on SF 1 by cross-linking the two reactive thiols, SH 1 and SH 2 , by N,N'-p-phenylene dimaleimide or Co(II)/Co(III) phenanthroline complexes. The Co(II)/Co(III) phenanthroline modified [ 14 C]Bz 2 ATP-SF 1 , after proteolytic digestion, yields five labeled peptides which were purified by gel filtration and high performance liquid chromatography

  11. The Binding Sites of miR-619-5p in the mRNAs of Human and Orthologous Genes.

    Science.gov (United States)

    Atambayeva, Shara; Niyazova, Raigul; Ivashchenko, Anatoliy; Pyrkova, Anna; Pinsky, Ilya; Akimniyazova, Aigul; Labeit, Siegfried

    2017-06-01

    Normally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs. This significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications. The search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. For the binding sites of the miR-619-5p the hybridization free energy of the bonds was equal to 100% of the maximum potential free energy. The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3'UTR (214 sites), CDS (3 sites), and 5'UTR (4 sites). The mRNAs of CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites, and the mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these miR-619-5p binding sites are located in the 3'UTRs. The miR-619-5p binding site in the 5'UTR of mRNA of human USP29 gene is found in the mRNAs of orthologous genes of primates. Binding sites of miR-619-5p in the coding regions of mRNAs of C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is present in the orthologous proteins. Binding sites of miR-619-5p in the mRNAs of transcription factor genes ZNF429 and ZNF429 encode the AHACNP oligopeptide in another reading frame. Binding sites of miR-619-5p in the 3'UTRs of all human target genes are also present in the 3'UTRs of orthologous genes of mammals. The completely complementary binding sites for miR-619-5p are conservative in the orthologous mammalian genes. The majority of miR-619-5p binding sites are located in the 3'UTRs but some genes have miRNA binding sites in the 5'UTRs of mRNAs. Several genes have binding sites for miRNAs in the CDSs that are read in different open reading frames. Identical nucleotide sequences of binding sites encode different amino acids in different proteins. The binding sites of miR-619-5p

  12. The peanut lectin-binding glycoproteins of human epidermal keratinocytes

    International Nuclear Information System (INIS)

    Morrison, A.I.; Keeble, S.; Watt, F.M.

    1988-01-01

    The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [ 14 C]galactose- or [ 14 C]mannose- and [ 14 C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification

  13. Thermodynamic parameters for binding of some halogenated inhibitors of human protein kinase CK2

    International Nuclear Information System (INIS)

    Winiewska, Maria; Makowska, Małgorzata; Maj, Piotr; Wielechowska, Monika; Bretner, Maria; Poznański, Jarosław; Shugar, David

    2015-01-01

    Highlights: • Two new compounds being potential human CK2a inhibitors are studied. • Their IC50 values were determined in vitro. • The heats of binding and kbind were estimated using DSC. • The increased stability of protein–ligand complexes was followed by fluorescence. • Methylated TBBt derivative (MeBr3Br) is almost as active as TBBt. - Abstract: The interaction of human CK2α with a series of tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) analogs, in which one of the bromine atoms proximal to the triazole/imidazole ring is replaced by a methyl group, was studied by biochemical (IC 50 ) and biophysical methods (thermal stability of protein–ligand complex monitored by DSC and fluorescence). Two newly synthesized tri-bromo derivatives display inhibitory activity comparable to that of the reference compounds, TBBt and TBBz, respectively. DSC analysis of the stability of protein–ligand complexes shows that the heat of ligand binding (H bind ) is driven by intermolecular electrostatic interactions involving the triazole/imidazole ring, as indicated by a strong correlation between H bind and ligand pK a . Screening, based on fluorescence-monitored thermal unfolding of protein–ligand complexes, gave comparable results, clearly identifying ligands that most strongly bind to the protein. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly, relative to possible intermolecular halogen bonding, in binding of the ligands to the CK2α ATP-binding site

  14. Thermodynamic parameters for binding of some halogenated inhibitors of human protein kinase CK2

    Energy Technology Data Exchange (ETDEWEB)

    Winiewska, Maria; Makowska, Małgorzata [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland); Maj, Piotr [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland); Nencki Institute of Experimental Biology PAS, Warszawa (Poland); Wielechowska, Monika; Bretner, Maria [Warsaw University of Technology, Faculty of Chemistry, Warszawa (Poland); Poznański, Jarosław, E-mail: jarek@ibb.waw.pl [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland); Shugar, David [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland)

    2015-01-02

    Highlights: • Two new compounds being potential human CK2a inhibitors are studied. • Their IC50 values were determined in vitro. • The heats of binding and kbind were estimated using DSC. • The increased stability of protein–ligand complexes was followed by fluorescence. • Methylated TBBt derivative (MeBr3Br) is almost as active as TBBt. - Abstract: The interaction of human CK2α with a series of tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) analogs, in which one of the bromine atoms proximal to the triazole/imidazole ring is replaced by a methyl group, was studied by biochemical (IC{sub 50}) and biophysical methods (thermal stability of protein–ligand complex monitored by DSC and fluorescence). Two newly synthesized tri-bromo derivatives display inhibitory activity comparable to that of the reference compounds, TBBt and TBBz, respectively. DSC analysis of the stability of protein–ligand complexes shows that the heat of ligand binding (H{sub bind}) is driven by intermolecular electrostatic interactions involving the triazole/imidazole ring, as indicated by a strong correlation between H{sub bind} and ligand pK{sub a}. Screening, based on fluorescence-monitored thermal unfolding of protein–ligand complexes, gave comparable results, clearly identifying ligands that most strongly bind to the protein. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly, relative to possible intermolecular halogen bonding, in binding of the ligands to the CK2α ATP-binding site.

  15. Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes.

    Science.gov (United States)

    Bončina, Matjaž; Podlipnik, Črtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

    2015-12-02

    Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Long chain fatty acids alter the interactive binding of ligands to the two principal drug binding sites of human serum albumin.

    Directory of Open Access Journals (Sweden)

    Keishi Yamasaki

    Full Text Available A wide variety of drugs bind to human serum albumin (HSA at its two principal sites, namely site I and site II. A number of reports indicate that drug binding to these two binding sites are not completely independent, and that interactions between ligands of these two discrete sites can play a role. In this study, the effect of the binding of long-chain fatty acids on the interactive binding between dansyl-L-asparagine (DNSA; site I ligand and ibuprofen (site II ligand at pH6.5 was examined. Binding experiments showed that the binding of sodium oleate (Ole to HSA induces conformational changes in the molecule, which, in turn, changes the individual binding of DNSA and ibuprofen, as well as the mode of interaction between these two ligands from a 'competitive-like' allosteric interaction in the case of the defatted HSA conformer to a 'nearly independent' binding in the case of non-defatted HSA conformer. Circular dichroism measurements indicated that ibuprofen and Ole are likely to modify the spatial orientation of DNSA at its binding site. Docking simulations suggest that the long-distance electric repulsion between DNSA and ibuprofen on defatted HSA contributes to a 'competitive-like' allosteric interaction, whereas extending the distance between ligands and/or increasing the flexibility or size of the DNSA binding site in fatted HSA evokes a change in the interaction mode to 'nearly independent' binding. The present findings provide further insights into the structural dynamics of HSA upon the binding of fatty acids, and its effects on drug binding and drug-drug interactions that occur on HSA.

  17. Isolation of low-molecular-weight lead-binding protein from human erythrocytes

    International Nuclear Information System (INIS)

    Raghavan, S.R.V.; Gonick, H.C.

    1977-01-01

    In blood, lead is mainly associated with erythrocytes and only a very small amount is found in plasma. Previously it was thought that the lead was bound to the erythrocyte cell membrane but more recently it has been observed that lead is bound primarily to the cell contents, ostensibly hemoglobin. In examining the lead-binding properties of normal human erythrocytes and those of lead-exposed industrial workers, we have found that, whereas lead binds only to hemoglobin in normal erythrocytes, there is also appreciable binding of lead to a low-molecular weight-protein in erythrocytes from lead-exposed workers. The synthesis of this protein may be induced by lead exposure. The 10,000 molecular weight protein may act as a storage site and mechanism for segregating lead in a non-toxic form

  18. Confinement Sensing and Signal Optimization via Piezo1/PKA and Myosin II Pathways

    Directory of Open Access Journals (Sweden)

    Wei-Chien Hung

    2016-05-01

    Full Text Available Summary: Cells adopt distinct signaling pathways to optimize cell locomotion in different physical microenvironments. However, the underlying mechanism that enables cells to sense and respond to physical confinement is unknown. Using microfabricated devices and substrate-printing methods along with FRET-based biosensors, we report that, as cells transition from unconfined to confined spaces, intracellular Ca2+ level is increased, leading to phosphodiesterase 1 (PDE1-dependent suppression of PKA activity. This Ca2+ elevation requires Piezo1, a stretch-activated cation channel. Moreover, differential regulation of PKA and cell stiffness in unconfined versus confined cells is abrogated by dual, but not individual, inhibition of Piezo1 and myosin II, indicating that these proteins can independently mediate confinement sensing. Signals activated by Piezo1 and myosin II in response to confinement both feed into a signaling circuit that optimizes cell motility. This study provides a mechanism by which confinement-induced signaling enables cells to sense and adapt to different physical microenvironments. : Hung et al. demonstrate that a Piezo1-dependent intracellular calcium increase negatively regulates protein kinase A (PKA as cells transit from unconfined to confined spaces. The Piezo1/PKA and myosin II signaling modules constitute two confinement-sensing mechanisms. This study provides a paradigm by which signaling enables cells to sense and adapt to different microenvironments.

  19. Probing the binding of fluoxetine hydrochloride to human serum albumin by multispectroscopic techniques

    Science.gov (United States)

    Katrahalli, Umesha; Jaldappagari, Seetharamappa; Kalanur, Shankara S.

    2010-01-01

    The interaction between human serum albumin (HSA) and fluoxetine hydrochloride (FLX) have been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, circular dichroism and FTIR under simulated physiological conditions. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of FLX to HSA. The values of binding constant, K of FLX-HSA were evaluated at 289, 300 and 310 K and were found to be 1.90 × 10 3, 1.68 × 10 3 and 1.45 × 10 3 M -1, respectively. The number of binding sites, n was noticed to be almost equal to unity thereby indicating the presence of a single class of binding site for FLX on HSA. Based on the thermodynamic parameters, Δ H0 and Δ S0 nature of binding forces operating between HSA and FLX were proposed. Spectral results revealed the conformational changes in protein upon interaction. Displacement studies indicated the site I as the main binding site for FLX on HSA. The effect of common ions on the binding of FLX to HSA was also investigated.

  20. Alterations in transcription factor binding in radioresistant human melanoma cells after ionizing radiation

    International Nuclear Information System (INIS)

    Sahijdak, W.M.; Yang, Chin-Rang; Zuckerman, J.S.; Meyers, M.; Boothman, D.A.

    1994-01-01

    We analyzed alterations in transcription factor binding to specific, known promoter DNA consensus sequences between irradiated and unirradiated radioresistant human melanoma (U1-Mel) cells. The goal of this study was to begin to investigate which transcription factors and DNA-binding sites are responsible for the induction of specific transcripts and proteins after ionizing radiation. Transcription factor binding was observed using DNA band-shift assays and oligonucleotide competition analyses. Confluence-arrested U1-Mel cells were irradiated (4.5 Gy) and harvested at 4 h. Double-stranded oligonucleotides containing known DNA-binding consensus sites for specific transcription factors were used. Increased DNA binding activity after ionizing radiation was noted with oligonucleotides containing the CREB, NF-kB and Sp1 consensus sites. No changes in protein binding to AP-1, AP-2, AP-3, or CTF/NF1, GRE or Oct-1 consensus sequences were noted. X-ray activation of select transcription factors, which bind certain consensus sites in promoters, may cause specific induction or repression of gene transcription. 22 refs., 2 figs

  1. Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism*

    Science.gov (United States)

    Gunawardane, Ruwanthi N.; Fordstrom, Preston; Piper, Derek E.; Masterman, Stephanie; Siu, Sophia; Liu, Dongming; Brown, Mike; Lu, Mei; Tang, Jie; Zhang, Richard; Cheng, Janet; Gates, Andrew; Meininger, David; Chan, Joyce; Carlson, Tim; Walker, Nigel; Schwarz, Margrit; Delaney, John; Zhou, Mingyue

    2016-01-01

    Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease. PMID:26644477

  2. The role of the Concanavalin A-binding fraction in cholesterol crystallization in native human bile

    NARCIS (Netherlands)

    Keulemans, Y. C.; Mok, K. S.; Gouma, D. J.; Groen, A. K.

    1997-01-01

    BACKGROUND/AIMS: Many Concanavalin A-binding glycoproteins have been proposed to influence cholesterol crystallization in human bile. This has been studied mainly by addition of the Concanavalin A-binding fraction to model bile. The physiological relevance of the proteins in native bile is not yet

  3. Lack of replication for the myosin-18B association with mathematical ability in independent cohorts

    Science.gov (United States)

    Pettigrew, K A; Fajutrao Valles, S F; Moll, K; Northstone, K; Ring, S; Pennell, C; Wang, C; Leavett, R; Hayiou-Thomas, M E; Thompson, P; Simpson, N H; Fisher, S E; Whitehouse, A J O; Snowling, M J; Newbury, D F; Paracchini, S

    2015-01-01

    Twin studies indicate that dyscalculia (or mathematical disability) is caused partly by a genetic component, which is yet to be understood at the molecular level. Recently, a coding variant (rs133885) in the myosin-18B gene was shown to be associated with mathematical abilities with a specific effect among children with dyslexia. This association represents one of the most significant genetic associations reported to date for mathematical abilities and the only one reaching genome-wide statistical significance. We conducted a replication study in different cohorts to assess the effect of rs133885 maths-related measures. The study was conducted primarily using the Avon Longitudinal Study of Parents and Children (ALSPAC), (N = 3819). We tested additional cohorts including the York Cohort, the Specific Language Impairment Consortium (SLIC) cohort and the Raine Cohort, and stratified them for a definition of dyslexia whenever possible. We did not observe any associations between rs133885 in myosin-18B and mathematical abilities among individuals with dyslexia or in the general population. Our results suggest that the myosin-18B variant is unlikely to be a main factor contributing to mathematical abilities. PMID:25778778

  4. The effect of the substitution of D{sub 2}O for H{sub 2}O on the degradation of myosin {beta} in solution by heat and by {sup 60}Co {gamma} radiation (1962); Effet de la substitution de D{sub 2}O a H{sub 2}O sur l'alteration de la Myosine B en solution par la chaleur et par les rayons {gamma} du {sup 60}CO (1962)

    Energy Technology Data Exchange (ETDEWEB)

    Pinset-Harstrom, I.; Fritsch, A. [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

    1962-07-01

    (1) Alterations of myosin B produced by heat or irradiation are shown to be qualitatively identical as demonstrated by analytical centrifugation. (2) A considerable isotope effect was demonstrated using 75 per cent D{sub 2}O in the solvent. The sensitivity of myosin B to heat and irradiation is discussed in the light of this isotope effect. (3) Polymers appearing upon heat treatment of myosin B seem to be of a very different nature than the polymers occurring alter a similar treatment upon myosin A. Polymers obtained from myosin B can be depolymerized by ATP and they appear in a much narrower temperature range than myosin A polymers. This fact indicates a considerable difference in the activation enthalpies in the two reactions. (authors) [French] (1) Cette etude montre que les alterations de la myosine B provoquees par la chaleur et par l'irradiation aux rayons {gamma} sont - telles qu'elles apparaissent a l'ultracentrifugation analytique - qualitativement semblables. (2) Nous avons observe un effet isotopique considerable de la presence de 75 pour cent de D{sub 2}O dans le solvant sur la sensibilite de la myosine B envers ces deux agents, et nous avons presente une tentative d'explication de ce fait. (3) Les polymeres qui apparaissent apres un traitement par la chaleur de la myosine semblent etre d'une nature tres differente des polymeres que l'on voit apparaitre apres un traitement identique de la myosine A. Ceux obtenus a partir de le myosine B sont depolymerisables par l'intermediaire de l'ATP et apparaissent dans une zone de temperature beaucoup plus etroite que celles de la myosine A. Ce dernier fait indique une difference considerable de l'enthalpie d'activation des deux reactions. (auteurs)

  5. The effect of the substitution of D{sub 2}O for H{sub 2}O on the degradation of myosin {beta} in solution by heat and by {sup 60}Co {gamma} radiation (1962); Effet de la substitution de D{sub 2}O a H{sub 2}O sur l'alteration de la Myosine B en solution par la chaleur et par les rayons {gamma} du {sup 60}CO (1962)

    Energy Technology Data Exchange (ETDEWEB)

    Pinset-Harstrom, I; Fritsch, A [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

    1962-07-01

    (1) Alterations of myosin B produced by heat or irradiation are shown to be qualitatively identical as demonstrated by analytical centrifugation. (2) A considerable isotope effect was demonstrated using 75 per cent D{sub 2}O in the solvent. The sensitivity of myosin B to heat and irradiation is discussed in the light of this isotope effect. (3) Polymers appearing upon heat treatment of myosin B seem to be of a very different nature than the polymers occurring alter a similar treatment upon myosin A. Polymers obtained from myosin B can be depolymerized by ATP and they appear in a much narrower temperature range than myosin A polymers. This fact indicates a considerable difference in the activation enthalpies in the two reactions. (authors) [French] (1) Cette etude montre que les alterations de la myosine B provoquees par la chaleur et par l'irradiation aux rayons {gamma} sont - telles qu'elles apparaissent a l'ultracentrifugation analytique - qualitativement semblables. (2) Nous avons observe un effet isotopique considerable de la presence de 75 pour cent de D{sub 2}O dans le solvant sur la sensibilite de la myosine B envers ces deux agents, et nous avons presente une tentative d'explication de ce fait. (3) Les polymeres qui apparaissent apres un traitement par la chaleur de la myosine semblent etre d'une nature tres differente des polymeres que l'on voit apparaitre apres un traitement identique de la myosine A. Ceux obtenus a partir de le myosine B sont depolymerisables par l'intermediaire de l'ATP et apparaissent dans une zone de temperature beaucoup plus etroite que celles de la myosine A. Ce dernier fait indique une difference considerable de l'enthalpie d'activation des deux reactions. (auteurs)

  6. Study of deutero-isotopomer-induced inhibition of caffeine and phenobarbitone binding to human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Cherrah, Y.; Falconnet, J.B.; Desage, M.; Brazier, J.L.; Zini, R.; Tillement, J.P.

    1988-01-01

    The present study of inhibition provides confirmation to previously observed deuterium isotope effects on in vitro caffeine and phenobarbitone binding to human serum albumin (HSA). Addition of either 3,7(C(/sup 2/H)/sub 3/)/sub 2/ or 1,3,7(C(/sup 2/H)/sub 3/)/sub 3/ caffeine induces a 50% loss in both the extent of binding and binding parameters of the unlabelled analog. As concerns caffeine displacement from its HSA sites, it is shown that phenobarbitone and its 5-pentadeuterophenyl analog are equally potent inhibitors of caffeine binding, though individual HSA binding profiles differ. As for HSA binding interactions between phenobarbitone isotopomers, a 50% decrease in unlabelled phenobarbitone extent of binding is observed in the presence of its 5-pentadeuterophenyl analog. Results favor the hypothesis of differing binding sites for each isotopomer.

  7. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasitePlasmodium knowlesi

    KAUST Repository

    Moon, Robert W.; Sharaf, Hazem; Hastings, Claire H.; Ho, Yung Shwen; Nair, Mridul; Rchiad, ‍ Zineb; Knuepfer, Ellen; Ramaprasad, Abhinay; Mohring, Franziska; Amir, Amirah; Yusuf, Noor A.; Hall, Joanna; Almond, Neil; Lau, Yee Ling; Pain, Arnab; Blackman, Michael J.; Holder, Anthony A.

    2016-01-01

    The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

  8. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasitePlasmodium knowlesi

    KAUST Repository

    Moon, Robert W.

    2016-06-15

    The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

  9. Myosin Va associates with mRNA in ribonucleoprotein particles present in myelinated peripheral axons and in the central nervous system.

    Science.gov (United States)

    Calliari, Aldo; Farías, Joaquina; Puppo, Agostina; Canclini, Lucía; Mercer, John A; Munroe, David; Sotelo, José R; Sotelo-Silveira, José R

    2014-03-01

    Sorting of specific mRNAs to particular cellular locations and regulation of their translation is an essential mechanism underlying cell polarization. The transport of RNAs by kinesins and dyneins has been clearly established in several cell models, including neurons in culture. A similar role appears to exist in higher eukaryotes for the myosins. Myosin Va (Myo5a) has been described as a component of ribonucleoprotein particles (RNPs) in the adult rat nervous system and associated to ZBP1 and ribosomes in ribosomal periaxoplasmic plaques (PARPs), making it a likely candidate for mediating some aspects of RNA transport in neurons. To test this hypothesis, we have characterized RNPs containing Myo5a in adult brains of rats and mice. Microarray analysis of RNAs co-immunoprecipitated with Myo5a indicates that this motor may associate with a specific subpopulation of neuronal mRNAs. We found mRNAs encoding α-synuclein and several proteins with functions in translation in these RNPs. Immunofluorescence analyses of RNPs showed apparent co-localization of Myo5a with ribosomes, mRNA and RNA-binding proteins in discrete structures present both in axons of neurons in culture and in myelinated fibers of medullary roots. Our data suggest that PARPs include RNPs bearing the mRNA coding for Myo5a and are equipped with kinesin and Myo5a molecular motors. In conclusion, we suggest that Myo5a is involved in mRNA trafficking both in the central and peripheral nervous systems. Copyright © 2013 Wiley Periodicals, Inc.

  10. The force dependence of isometric and concentric potentiation in mouse muscle with and without skeletal myosin light chain kinase.

    Science.gov (United States)

    Gittings, William; Aggarwal, Harish; Stull, James T; Vandenboom, Rene

    2015-01-01

    The isometric potentiation associated with myosin phosphorylation is force dependent. The purpose of this study was to assess the influence of a pre-existing period of isometric force on the concentric force potentiation displayed by mouse muscles with and without the ability to phosphorylate myosin. We tested isometric (ISO) and concentric (CON) potentiation, as well as concentric potentiation after isometric force (ISO-CON), in muscles from wild-type (WT) and skeletal myosin light chain kinase-deficient (skMLCK(-/-)) mice. A conditioning stimulus increased (i.e., potentiated) mean concentric force in the ISO-CON and CON conditions to 1.31 ± 0.02 and 1.35 ± 0.02 (WT) and to 1.19 ± 0.02 and 1.21 ± 0.01 (skMLCK(-/-)) of prestimulus levels, respectively (data n = 6-8, p muscles.

  11. Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion.

    Science.gov (United States)

    Scott, Kevin G-E; Meddings, Jonathon B; Kirk, David R; Lees-Miller, Susan P; Buret, André G

    2002-10-01

    Giardiasis causes malabsorptive diarrhea, and symptoms can be present in the absence of any significant morphologic injury to the intestinal mucosa. The effects of giardiasis on epithelial permeability in vivo remain unknown, and the role of T cells and myosin light chain kinase (MLCK) in altered intestinal barrier function is unclear. This study was conducted to determine whether Giardia spp. alters intestinal permeability in vivo, to assess whether these abnormalities are dependent on T cells, and to assess the role of MLCK in altered epithelial barrier function. Immunocompetent and isogenic athymic mice were inoculated with axenic Giardia muris trophozoites or sterile vehicle (control), then assessed for trophozoite colonization and gastrointestinal permeability. Mechanistic studies using nontransformed human duodenal epithelial monolayers (SCBN) determined the effects of Giardia on myosin light chain (MLC) phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, cytoskeletal F-actin, tight junctional zonula occludens-1 (ZO-1), and MLCK. Giardia infection caused a significant increase in small intestinal, but not gastric or colonic, permeability that correlated with trophozoite colonization in both immunocompetent and athymic mice. In vitro, Giardia increased permeability and phosphorylation of MLC and reorganized F-actin and ZO-1. These alterations were abolished with an MLCK inhibitor. Disruption of small intestinal barrier function is T cell independent, disappears on parasite clearance, and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1 in an MLCK-dependent fashion.

  12. Expression of porcine myosin heavy chain 1 gene in Berkshire loins ...

    African Journals Online (AJOL)

    Expression of porcine myosin heavy chain 1 gene in Berkshire loins with a high pH24 value. Jin Hun Kang, Woo Young Bang, Eun Jung Kwon, Yong Hwa Lee, Da Hye Park, Eun Seok Cho, Min Ji Kim, Jong-Soon Choi, Hwa Chun Park, Beom Young Park, Chul Wook Kim ...

  13. Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S., E-mail: bcssj@iacs.res.in

    2014-08-01

    3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.

  14. MicroRNA-363 targets myosin 1B to reduce cellular migration in head and neck cancer

    International Nuclear Information System (INIS)

    Chapman, Bhavana V.; Wald, Abigail I.; Akhtar, Parvez; Munko, Ana C.; Xu, Jingjing; Gibson, Sandra P.; Grandis, Jennifer R.; Ferris, Robert L.; Khan, Saleem A.

    2015-01-01

    Squamous cell carcinoma of the head and neck (SCCHN) remains a prevalent and devastating disease. Recently, there has been an increase in SCCHN cases that are associated with high-risk human papillomavirus (HPV) infection. The clinical characteristics of HPV-positive and HPV-negative SCCHN are known to be different but their molecular features are only recently beginning to emerge. MicroRNAs (miRNAs, miRs) are small, non-coding RNAs that are likely to play significant roles in cancer initiation and progression where they may act as oncogenes or tumor suppressors. Previous studies in our laboratory showed that miR-363 is overexpressed in HPV-positive compared to HPV-negative SCCHN cell lines, and the HPV type 16-E6 oncoprotein upregulates miR-363 in SCCHN cell lines. However, the functional role of miR-363 in SCCHN in the context of HPV infection remains to be elucidated. We analyzed miR-363 levels in SCCHN tumors with known HPV-status from The Cancer Genome Atlas (TCGA) and an independent cohort from our institution. Cell migration studies were conducted following the overexpression of miR-363 in HPV-negative cell lines. Bioinformatic tools and a luciferase reporter assay were utilized to confirm that miR-363 targets the 3’-UTR of myosin 1B (MYO1B). MYO1B mRNA and protein expression levels were evaluated following miR-363 overexpression in HPV-negative SCCHN cell lines. Small interfering RNA (siRNA) knockdown of MYO1B was performed to assess the phenotypic implication of reduced MYO1B expression in SCCHN cell lines. MiR-363 was found to be overexpressed in HPV-16-positive compared to the HPV-negative SCCHN tumors. Luciferase reporter assays performed in HPV-negative JHU028 cells confirmed that miR-363 targets one of its two potential binding sites in the 3’UTR of MYO1B. MYO1B mRNA and protein levels were reduced upon miR-363 overexpression in four HPV-negative SCCHN cell lines. Increased miR-363 expression or siRNA knockdown of MYO1B expression reduced

  15. Structures of the Streptococcus sanguinis SrpA Binding Region with Human Sialoglycans Suggest Features of the Physiological Ligand.

    Science.gov (United States)

    Loukachevitch, Lioudmila V; Bensing, Barbara A; Yu, Hai; Zeng, Jie; Chen, Xi; Sullam, Paul M; Iverson, T M

    2016-10-11

    Streptococcus sanguinis is a leading cause of bacterial infective endocarditis, a life-threatening infection of heart valves. S. sanguinis binds to human platelets with high avidity, and this adherence is likely to enhance virulence. Previous studies suggest that a serine-rich repeat adhesin termed SrpA mediates the binding of S. sanguinis to human platelets via its interaction with sialoglycans on the receptor GPIbα. However, in vitro binding assays with SrpA and defined sialoglycans failed to identify specific high-affinity ligands. To improve our understanding of the interaction between SrpA and human platelets, we determined cocrystal structures of the SrpA sialoglycan binding region (SrpA BR ) with five low-affinity ligands: three sialylated trisaccharides (sialyl-T antigen, 3'-sialyllactose, and 3'-sialyl-N-acetyllactosamine), a sialylated tetrasaccharide (sialyl-Lewis X ), and a sialyl galactose disaccharide component common to these sialoglyans. We then combined structural analysis with mutagenesis to further determine whether our observed interactions between SrpA BR and glycans are important for binding to platelets and to better map the binding site for the physiological receptor. We found that the sialoglycan binding site of SrpA BR is significantly larger than the sialoglycans cocrystallized in this study, which suggests that binding of SrpA to platelets either is multivalent or occurs via a larger, disialylated glycan.

  16. Exogenous fatty acid binding protein 4 promotes human prostate cancer cell progression.

    Science.gov (United States)

    Uehara, Hisanori; Takahashi, Tetsuyuki; Oha, Mina; Ogawa, Hirohisa; Izumi, Keisuke

    2014-12-01

    Epidemiologic studies have found that obesity is associated with malignant grade and mortality in prostate cancer. Several adipokines have been implicated as putative mediating factors between obesity and prostate cancer. Fatty acid binding protein 4 (FABP4), a member of the cytoplasmic fatty acid binding protein multigene family, was recently identified as a novel adipokine. Although FABP4 is released from adipocytes and mean circulating concentrations of FABP4 are linked with obesity, effects of exogenous FABP4 on prostate cancer progression are unclear. In this study, we examined the effects of exogenous FABP4 on human prostate cancer cell progression. FABP4 treatment promoted serum-induced prostate cancer cell invasion in vitro. Furthermore, oleic acid promoted prostate cancer cell invasion only if FABP4 was present in the medium. These promoting effects were reduced by FABP4 inhibitor, which inhibits FABP4 binding to fatty acids. Immunostaining for FABP4 showed that exogenous FABP4 was taken up into DU145 cells in three-dimensional culture. In mice, treatment with FABP4 inhibitor reduced the subcutaneous growth and lung metastasis of prostate cancer cells. Immunohistochemical analysis showed that the number of apoptotic cells, positive for cleaved caspase-3 and cleaved PARP, was increased in subcutaneous tumors of FABP4 inhibitor-treated mice, as compared with control mice. These results suggest that exogenous FABP4 might promote human prostate cancer cell progression by binding with fatty acids. Additionally, exogenous FABP4 activated the PI3K/Akt pathway, independently of binding to fatty acids. Thus, FABP4 might be a key molecule to understand the mechanisms underlying the obesity-prostate cancer progression link. © 2014 UICC.

  17. Manganese binding proteins in human and cow's milk

    International Nuclear Information System (INIS)

    Loennerdal, B.; Keen, C.L.; Hurley, L.S.

    1985-01-01

    Manganese nutrition in the neonatal period is poorly understood, due in part to a lack of information on the amount of manganese in infant foods and its bioavailability. Since the molecular localization of an element in foods is one determinant of its subsequent bioavailability, a study was made of the binding of manganese in human and cow's milk. An extrinsic label of 54 Mn was shown to equilibrate isotopically with native manganese in milks and formulas. Milk samples were separated into fat, casein and whey by ultracentrifugation. In human milk, the major part (71%) of manganese was found in whey, 11% in casein and 18% in the lipid fraction. In contrast, in cow's milk, 32% of total manganese was in whey, 67% in casein and 1% in lipid. Within the human whey fraction, most of the manganese was bound to lactoferrin, while in cow's whey, manganese was mostly complexed to ligands with molecular weights less than 200. The distribution of manganese in formulas was closer to that of human milk than of cow's milk. The bioavailability of manganese associated with lactoferrin, casein and low molecular weight complexes needs to be assessed

  18. Alteration of human serum albumin binding properties induced by modifications: A review

    Science.gov (United States)

    Maciążek-Jurczyk, Małgorzata; Szkudlarek, Agnieszka; Chudzik, Mariola; Pożycka, Jadwiga; Sułkowska, Anna

    2018-01-01

    Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlow's site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

  19. Acceleration of the sliding movement of actin filaments with the use of a non-motile mutant myosin in in vitro motility assays driven by skeletal muscle heavy meromyosin.

    Directory of Open Access Journals (Sweden)

    Kohei Iwase

    Full Text Available We examined the movement of an actin filament sliding on a mixture of normal and genetically modified myosin molecules that were attached to a glass surface. For this purpose, we used a Dictyostelium G680V mutant myosin II whose release rates of Pi and ADP were highly suppressed relative to normal myosin, leading to a significantly extended life-time of the strongly bound state with actin and virtually no motility. When the mixing ratio of G680V mutant myosin II to skeletal muscle HMM (heavy myosin was 0.01%, the actin filaments moved intermittently. When they moved, their sliding velocities were about two-fold faster than the velocity of skeletal HMM alone. Furthermore, sliding movements were also faster when the actin filaments were allowed to slide on skeletal muscle HMM-coated glass surfaces in the motility buffer solution containing G680V HMM. In this case no intermittent movement was observed. When the actin filaments used were copolymerized with a fusion protein consisting of Dictyostelium actin and Dictyostelium G680V myosin II motor domain, similar faster sliding movements were observed on skeletal muscle HMM-coated surfaces. The filament sliding velocities were about two-fold greater than the velocities of normal actin filaments. We found that the velocity of actin filaments sliding on skeletal muscle myosin molecules increased in the presence of a non-motile G680V mutant myosin motor.

  20. Parathyroid hormone promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: Mediation by cyclic AMP

    International Nuclear Information System (INIS)

    Egan, J.J.; Gronowicz, G.; Rodan, G.A.

    1991-01-01

    Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X-100 (1%) nonextractable proteins. After 2-5 minutes, PTH caused a rapid and transient decrease of 50-60% in polymerized actin and myosin associated with the Triton X-100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose-dependent with an IC50 of about 1 nM, and was partially inhibited by the (3-34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin-2/AM (10 microM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (PI 4.9) myosin light chain in cells preincubated with [32P]-orthophosphate. The phosphorylation of this protein decreased within 2-3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A-23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine-conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP

  1. Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

    International Nuclear Information System (INIS)

    Schmitz, G.; Wulf, G.; Bruening, T.A.; Assmann, G.

    1987-01-01

    In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125 I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 x 10(4)/cell), followed by monocytes (7.3 x 10(4)/cell) and lymphocytes (4.0 x 10(4)/cell). Compared with conventional analysis of binding of 125 I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125 I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a docking type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias

  2. Blebbistain, a myosin II inhibitor, as a novel strategy to regulate detrusor contractility in a rat model of partial bladder outlet obstruction.

    Directory of Open Access Journals (Sweden)

    Xinhua Zhang

    Full Text Available Partial bladder outlet obstruction (PBOO, a common urologic pathology mostly caused by benign prostatic hyperplasia, can coexist in 40-45% of patients with overactive bladder (OAB and is associated with detrusor overactivity (DO. PBOO that induces DO results in alteration in bladder myosin II type and isoform composition. Blebbistatin (BLEB is a myosin II inhibitor we recently demonstrated potently relaxed normal detrusor smooth muscle (SM and reports suggest varied BLEB efficacy for different SM myosin (SMM isoforms and/or SMM vs nonmuscle myosin (NMM. We hypothesize BLEB inhibition of myosin II as a novel contraction protein targeted strategy to regulate DO. Using a surgically-induced male rat PBOO model, organ bath contractility, competitive and Real-Time-RT-PCR were performed. It was found that obstructed-bladder weight significantly increased 2.74-fold while in vitro contractility of detrusor to various stimuli was impaired ∼50% along with decreased shortening velocity. Obstruction also altered detrusor spontaneous activities with significantly increased amplitude but depressed frequency. PBOO switched bladder from a phasic-type to a more tonic-type SM. Expression of 5' myosin heavy chain (MHC alternatively spliced isoform SM-A (associated with tonic-type SM increased 3-fold while 3' MHC SM1 and essential light chain isoform MLC(17b also exhibited increased relative expression. Total SMMHC expression was decreased by 25% while the expression of NMM IIB (SMemb was greatly increased by 4.5-fold. BLEB was found to completely relax detrusor strips from both sham-operated and PBOO rats pre-contracted with KCl, carbachol or electrical field stimulation although sensitivity was slightly decreased (20% only at lower doses for PBOO. Thus we provide the first thorough characterization of the response of rat bladder myosin to PBOO and demonstrate complete BLEB-induced PBOO bladder SM relaxation. Furthermore, the present study provides valuable

  3. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    Energy Technology Data Exchange (ETDEWEB)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany); Renkawitz-Pohl, Renate, E-mail: renkawit@biologie.uni-marburg.de [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany)

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  4. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    International Nuclear Information System (INIS)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev; Renkawitz-Pohl, Renate

    2013-01-01

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity

  5. Potential toxicity of sulfanilamide antibiotic: Binding of sulfamethazine to human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jiabin [State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092 (China); Zhou, Xuefei [Key Laboratory of Yangtze River Water Environment for Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092 (China); Zhang, Yalei, E-mail: zhangyalei2003@163.com [State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092 (China); Gao, Haiping [Key Laboratory of Yangtze River Water Environment for Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092 (China)

    2012-08-15

    Antibiotics are widely used in daily life but their abuse has posed a potential threat to human health. The interaction between human serum albumin (HSA) and sulfamethazine (SMZ) was investigated by capillary electrophoresis, fluorescence spectrometry, and circular dichroism. The binding constant and site were determined to be 1.09 Multiplication-Sign 10{sup 4} M{sup -1} and 1.14 at 309.5 K. The thermodynamic determination indicated that the interaction was driven by enthalpy change, where the electrostatic interaction and hydrogen bond were the dominant binding force. The binding distance between SMZ and tryptophan residue of HSA was obtained to be 3.07 nm according to Foerster non-radioactive energy transfer theory. The site marker competition revealed that SMZ bound into subdomain IIA of HSA. The binding of SMZ induced the unfolding of the polypeptides of HSA and transferred the secondary conformation of HSA. The equilibrium dialysis showed that only 0.13 mM SMZ decreased vitamin B{sub 2} by 38% transported on the HSA. This work provides a new quantitative evaluation method for antibiotics to cause the protein damage. -- Highlights: Black-Right-Pointing-Pointer Various techniques characterized the interactions between SMZ and HSA. Black-Right-Pointing-Pointer The electrostatic interaction and hydrogen bond dominated in the interaction. Black-Right-Pointing-Pointer SMZ induced the conformation change of HSA. Black-Right-Pointing-Pointer SMZ affected the transportation function of HSA.

  6. Structure of Calmodulin Bound to a Calcineurin Peptide: A New Way of Making an Old Binding Mode

    International Nuclear Information System (INIS)

    Ye, Q.; Li, X.; Wong, A.; Wei, Q.; Jia, Z.

    2006-01-01

    Calcineurin is a calmodulin-binding protein in brain and the only serine/threonine protein phosphatase under the control of Ca 2+ /calmodulin (CaM), which plays a critical role in coupling Ca 2+ signals to cellular responses. CaM up-regulates the phosphatase activity of calcineurin by binding to the CaM-binding domain (CBD) of calcineurin subunit A. Here, we report crystal structural studies of CaM bound to a CBD peptide. The chimeric protein containing CaM and the CBD peptide forms an intimate homodimer, in which CaM displays a native-like extended conformation and the CBD peptide shows -helical structure. Unexpectedly, the N-terminal lobe from one CaM and the C-terminal lobe from the second molecule form a combined binding site to trap the peptide. Thus, the dimer provides two binding sites, each of which is reminiscent of the fully collapsed conformation of CaM commonly observed in complex with, for example, the myosin light chain kinase (MLCK) peptide. The interaction between the peptide and CaM is highly specific and similar to MLCK

  7. Water binding of proteins in the processing frankfurter-type sausages. Part. 1. Water-binding ability of freeze-dried meat fractions containing myofibrillar and stromal proteins.

    Science.gov (United States)

    Heinevetter, L; Gassmann, B; Kroll, J

    1987-01-01

    As soon as possible and 48 h after slaughter respectively, from both blade-bone muscle groups of cattle and pig carcasses the "thick pieces" were excised, extracted, and fractionated. Residues and precipitates from water and salt extracts resulted were freeze-dried, and an improved Baumann capillary suction apparatus was used to measure their water binding capacity (WBC) with and without addition of 2% sodium chloride and/or heating to 80 degrees C. With one exception the WBC results followed a relative pattern demonstrating the final residues (stromal proteins and leavings of myofibrillar proteins) binding the highest amount of added water, precipitates of dialysis (mainly containing myofibrillar proteins) a remarkable amount and powdered meats the least. As scanning electron micrographs confirmed, there were no fibrous structures in the precipitates resulted from dialysis of salt solutions (1.0 mol/1). Heating decreased the spontaneous water uptake of all fractions. Addition of sodium chloride had only a noticeable capillary-suction and swelling effect on unheated samples. Hence swelling of undissolved protein structures (extraction of myosin and possibly of actomyosin) is therefore not the only way for water binding in frankfurter-type sausages.

  8. Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function

    Directory of Open Access Journals (Sweden)

    Matthew J. Birket

    2015-10-01

    Full Text Available Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC model of hypertrophic cardiomyopathy (HCM. A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

  9. Human liver aldehyde dehydrogenase: coenzyme binding

    International Nuclear Information System (INIS)

    Kosley, L.L.; Pietruszko, R.

    1987-01-01

    The binding of [U- 14 C] NAD to mitochondrial (E2) and cytoplasmin(E1) aldehyde dehydrogenase was measured by gel filtration and sedimentation techniques. The binding data for NAD and (E1) yielded linear Scatchard plots giving a dissociation constant of 25 (+/- 8) uM and the stoichiometry of 2 mol of NAD bound per mol of E1. The binding data for NAD and (E2) gave nonlinear Scatchard plots. The binding of NADH to E2 was measured via fluorescence enhancement; this could not be done with E1 because there was no signal. The dissociation constant for E2 by this technique was 0.7 (+/- 0.4) uM and stoichiometry of 1.0 was obtained. The binding of [U- 14 C] NADH to (E1) and (E2) was also measured by the sedimentation technique. The binding data for (E1) and NADH gave linear Scatchard plots giving a dissociation constant of 13 (+/- 6) uM and the stoichiometry of 2.0. The binding data for NADH to (E2) gave nonlinear Scatchard plots. With (E1), the dissociation constants for both NAD and NADH are similar to those determined kinetically, but the stoichiometry is only half of that found by stopped flow technique. With (E2) the dissociation constant by fluorometric procedure was 2 orders of magnitude less than that from catalytic reaction

  10. Nonmuscle myosin heavy chain IIA is a critical factor contributing to the efficiency of early infection of severe fever with thrombocytopenia syndrome virus.

    Science.gov (United States)

    Sun, Yinyan; Qi, Yonghe; Liu, Chenxuan; Gao, Wenqing; Chen, Pan; Fu, Liran; Peng, Bo; Wang, Haimin; Jing, Zhiyi; Zhong, Guocai; Li, Wenhui

    2014-01-01

    Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus in the Bunyaviridae family. Most patients infected by SFTSV present with fever and thrombocytopenia, and up to 30% die due to multiple-organ dysfunction. The mechanisms by which SFTSV enters multiple cell types are unknown. SFTSV contains two species of envelope glycoproteins, Gn (44.2 kDa) and Gc (56 kDa), both of which are encoded by the M segment and are cleaved from a precursor polypeptide (about 116 kDa) in the endoplasmic reticulum (ER). Gn fused with an immunoglobulin Fc tag at its C terminus (Gn-Fc) bound to multiple cells susceptible to the infection of SFTSV and blocked viral infection of human umbilical vein endothelial cells (HUVECs). Immunoprecipitation assays following mass spectrometry analysis showed that Gn binds to nonmuscle myosin heavy chain IIA (NMMHC-IIA), a cellular protein with surface expression in multiple cell types. Small interfering RNA (siRNA) knockdown of NMMHC-IIA, but not the closely related NMMHC-IIB or NMMHC-IIC, reduced SFTSV infection, and NMMHC-IIA specific antibody blocked infection by SFTSV but not other control viruses. Overexpression of NMMHC-IIA in HeLa cells, which show limited susceptivity to SFTSV, markedly enhanced SFTSV infection of the cells. These results show that NMMHC-IIA is critical for the cellular entry of SFTSV. As NMMHC-IIA is essential for the normal functions of platelets and human vascular endothelial cells, it is conceivable that NMMHC-IIA directly contributes to the pathogenesis of SFTSV and may be a useful target for antiviral interventions against the viral infection.

  11. Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

    Directory of Open Access Journals (Sweden)

    D.V. Vassallo

    2008-09-01

    Full Text Available Lead (Pb2+ poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM to the bath. Changes in rate of stimulation (0.1-1.5 Hz, relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM, and the effect of isoproterenol (20 ng/mL were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

  12. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

    2015-01-01

    Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells ofte...... or proteome using human leukocyte antigen binding predictions and made a web-accessible software implementation freely available at http://met-hilab.cbs.dtu.dk/blockcons/....

  13. Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.

    Science.gov (United States)

    Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

    2007-05-01

    The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.

  14. Myosin II activity is required for functional leading-edge cells and closure of epidermal sheets in fish skin ex vivo.

    Science.gov (United States)

    Morita, Toshiyuki; Tsuchiya, Akiko; Sugimoto, Masazumi

    2011-09-01

    Re-epithelialization in skin wound healing is a process in which epidermal sheets grow and close the wound. Although the actin-myosin system is thought to have a pivotal role in re-epithelialization, its role is not clear. In fish skin, re-epithelialization occurs around 500 μm/h and is 50 times faster than in mammalian skin. We had previously reported that leading-edge cells of the epidermal outgrowth have both polarized large lamellipodia and "purse string"-like actin filament cables in the scale-skin culture system of medaka fish, Oryzias latipes (Cell Tissue Res, 2007). The actin purse-string (APS) is a supracellular contractile machinery in which adherens junctions (AJs) link intracellular myosin II-including actin cables between neighboring cells. In this study, we developed a modified "face-to-face" scale-skin culture system as an ex vivo model to study epidermal wound healing, and examined the role of the actin-myosin system in the rapid re-epithelialization using a myosin II ATPase inhibitor, blebbistatin. A low level of blebbistatin suppressed the formation of APS and induced the dissociation of keratocytes from the leading edge without attenuating the growth of the epidermal sheet or the migration rate of solitary keratocytes. AJs in the superficial layer showed no obvious changes elicited by blebbistatin. However, two epidermal sheets without APSs did not make a closure with each other, which was confirmed by inhibiting the connecting AJs between the superficial layers. These results suggest that myosin II activity is required for functional leading-edge cells and for epidermal closure.

  15. Vitamin B12 Phosphate Conjugation and Its Effect on Binding to the Human B12 -Binding Proteins Intrinsic Factor and Haptocorrin

    DEFF Research Database (Denmark)

    Ó Proinsias, Keith; Ociepa, Michał; Pluta, Katarzyna

    2016-01-01

    The binding of vitamin B12 derivatives to human B12 transporter proteins is strongly influenced by the type and site of modification of the cobalamin original structure. We have prepared the first cobalamin derivative modified at the phosphate moiety. The reaction conditions were fully optimized...... and its limitations examined. The resulting derivatives, particularly those bearing terminal alkyne and azide groups, were isolated and used in copper-catalyzed alkyne-azide cycloaddition reactions (CuAAC). Their sensitivity towards light revealed their potential as photocleavable molecules. The binding...... abilities of selected derivatives were examined and compared with cyanocobalamin. The interaction of the alkylated derivatives with haptocorrin was less affected than the interaction with intrinsic factor. Furthermore, the configuration of the phosphate moiety was irrelevant to the binding process....

  16. Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

    International Nuclear Information System (INIS)

    Ritvos, O.; Ranta, T.; Jalkanen, J.; Suikkari, A.M.; Voutilainen, R.; Bohn, H.; Rutanen, E.M.

    1988-01-01

    The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purified 34 K IGF-BP specifically bound [125I]iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of [125I] iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of [125I] iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with [125I]iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-[3H]aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells

  17. Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics

    Directory of Open Access Journals (Sweden)

    Gowda Veerabasappa T

    2007-12-01

    Full Text Available Abstract Background The snake venom group IIA secreted phospholipases A2 (SVPLA2, present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL at the membrane/water interface and by highly specific direct binding to: (i presynaptic membrane-bound or intracellular receptors; (ii natural PLA2-inhibitors from snake serum; and (iii coagulation factors present in human blood. Results Using surface plasmon resonance (SPR protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS. The interface in FXa involves both, the light and heavy chains. Conclusion We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors.

  18. Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport

    NARCIS (Netherlands)

    Stapleton, Nigel M.; Armstrong-Fisher, Sylvia S.; Andersen, Jan Terje; van der Schoot, C. Ellen; Porter, Charlene; Page, Kenneth R.; Falconer, Donald; de Haas, Masja; Williamson, Lorna M.; Clark, Michael R.; Vidarsson, Gestur; Armour, Kathryn L.

    2018-01-01

    We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the

  19. Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor.

    Science.gov (United States)

    Maillet, Emeline L; Cui, Meng; Jiang, Peihua; Mezei, Mihaly; Hecht, Elizabeth; Quijada, Jeniffer; Margolskee, Robert F; Osman, Roman; Max, Marianna

    2015-10-01

    The sweet taste receptor, a heterodimeric G protein-coupled receptor comprised of T1R2 and T1R3, binds sugars, small molecule sweeteners, and sweet proteins to multiple binding sites. The dipeptide sweetener, aspartame binds in the Venus Flytrap Module (VFTM) of T1R2. We developed homology models of the open and closed forms of human T1R2 and human T1R3 VFTMs and their dimers and then docked aspartame into the closed form of T1R2's VFTM. To test and refine the predictions of our model, we mutated various T1R2 VFTM residues, assayed activity of the mutants and identified 11 critical residues (S40, Y103, D142, S144, S165, S168, Y215, D278, E302, D307, and R383) in and proximal to the binding pocket of the sweet taste receptor that are important for ligand recognition and activity of aspartame. Furthermore, we propose that binding is dependent on 2 water molecules situated in the ligand pocket that bridge 2 carbonyl groups of aspartame to residues D142 and L279. These results shed light on the activation mechanism and how signal transmission arising from the extracellular domain of the T1R2 monomer of the sweet receptor leads to the perception of sweet taste. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Lack of replication for the myosin-18B association with mathematical ability in independent cohorts.

    Science.gov (United States)

    Pettigrew, K A; Fajutrao Valles, S F; Moll, K; Northstone, K; Ring, S; Pennell, C; Wang, C; Leavett, R; Hayiou-Thomas, M E; Thompson, P; Simpson, N H; Fisher, S E; Whitehouse, A J O; Snowling, M J; Newbury, D F; Paracchini, S

    2015-04-01

    Twin studies indicate that dyscalculia (or mathematical disability) is caused partly by a genetic component, which is yet to be understood at the molecular level. Recently, a coding variant (rs133885) in the myosin-18B gene was shown to be associated with mathematical abilities with a specific effect among children with dyslexia. This association represents one of the most significant genetic associations reported to date for mathematical abilities and the only one reaching genome-wide statistical significance. We conducted a replication study in different cohorts to assess the effect of rs133885 maths-related measures. The study was conducted primarily using the Avon Longitudinal Study of Parents and Children (ALSPAC), (N = 3819). We tested additional cohorts including the York Cohort, the Specific Language Impairment Consortium (SLIC) cohort and the Raine Cohort, and stratified them for a definition of dyslexia whenever possible. We did not observe any associations between rs133885 in myosin-18B and mathematical abilities among individuals with dyslexia or in the general population. Our results suggest that the myosin-18B variant is unlikely to be a main factor contributing to mathematical abilities. © 2015 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.

  1. Double phosphorylation of the myosin regulatory light chain during rigor mortis of bovine Longissimus muscle.

    Science.gov (United States)

    Muroya, Susumu; Ohnishi-Kameyama, Mayumi; Oe, Mika; Nakajima, Ikuyo; Shibata, Masahiro; Chikuni, Koichi

    2007-05-16

    To investigate changes in myosin light chains (MyLCs) during postmortem aging of the bovine longissimus muscle, we performed two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results of fluorescent differential gel electrophoresis showed that two spots of the myosin regulatory light chain (MyLC2) at pI values of 4.6 and 4.7 shifted toward those at pI values of 4.5 and 4.6, respectively, by 24 h postmortem when rigor mortis was completed. Meanwhile, the MyLC1 and MyLC3 spots did not change during the 14 days postmortem. Phosphoprotein-specific staining of the gels demonstrated that the MyLC2 proteins at pI values of 4.5 and 4.6 were phosphorylated. Furthermore, possible N-terminal region peptides containing one and two phosphoserine residues were detected in each mass spectrum of the MyLC2 spots at pI values of 4.5 and 4.6, respectively. These results demonstrated that MyLC2 became doubly phosphorylated during rigor formation of the bovine longissimus, suggesting involvement of the MyLC2 phosphorylation in the progress of beef rigor mortis. Bovine; myosin regulatory light chain (RLC, MyLC2); phosphorylation; rigor mortis; skeletal muscle.

  2. Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding.

    Science.gov (United States)

    Shi, Shuiliang; Kelly, Brian J; Wang, Congrong; Klingler, Ken; Chan, Albert; Eckert, George J; Trippel, Stephen B

    2018-03-01

    Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. Human IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. Secreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. The biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. These data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Porphyromonas endodontalis binds, reduces and grows on human hemoglobin.

    Science.gov (United States)

    Zerr, M; Drake, D; Johnson, W; Cox, C D

    2001-08-01

    Porphyromonas endodontalis is a black-pigmented, obligate anaerobic rod-shaped bacterium implicated as playing a major role in endodontic infections. We have previously shown that P. endodontalis requires the porphyrin nucleus, preferably supplied as hemoglobin, as a growth supplement. The bacteria also actively transport free iron, although this activity does not support growth in the absence of a porphyrin source. The purpose of this study was to further investigate the binding and subsequent utilization of human hemoglobin by P. endodontalis. P. endodontalis binds hemoglobin and reduces the Fe(III) porphyrin, resulting in a steady accumulation of ferrous hemoglobin. Reduction of methemoglobin was similar to the extracellular reduction of nitrobluetetrazolium in the presence of oxidizable substrate. Turbidimetric and viable cell determinations showed that P. endodontalis grew when supplied only hemoglobin. Therefore, we conclude that hemoglobin appears to serve as a sole carbon and nitrogen source, and that these bacteria reduce extracellular compounds at the expense of oxidized substrates.

  4. Interactions of Poly(amidoamine) Dendrimers with Human Serum Albumin: Binding Constants and Mechanisms

    OpenAIRE

    Giri, Jyotsnendu; Diallo, Mamadou S.; Simpson, André J.; Liu, Yi; Goddard, William A., III; Kumar, Rajeev; Woods, Gwen C.

    2011-01-01

    The interactions of nanomaterials with plasma proteins have a significant impact on their in vivo transport and fate in biological fluids. This article discusses the binding of human serum albumin (HSA) to poly(amidoamine) [PAMAM] dendrimers. We use protein-coated silica particles to measure the HSA binding constants (K_b) of a homologous series of 19 PAMAM dendrimers in aqueous solutions at physiological pH (7.4) as a function of dendrimer generation, terminal group, and core chemistry. To g...

  5. Characterizing the binding motifs of 11 common human HLA‐DP and HLA‐DQ molecules using NNAlign

    DEFF Research Database (Denmark)

    Andreatta, Massimo; Nielsen, Morten

    2012-01-01

    ‐based method NNAlign, we characterized the binding specificities of five HLA‐DP and six HLA‐DQ among the most frequent in the human population. The identified binding motifs showed an overall concurrence with earlier studies but revealed subtle differences. The DP molecules revealed a large overlap...

  6. Formation and characterization of iron-binding phosphorylated human-like collagen as a potential iron supplement

    International Nuclear Information System (INIS)

    Deng, Jianjun; Chen, Fei; Fan, Daidi; Zhu, Chenhui; Ma, Xiaoxuan; Xue, Wenjiao

    2013-01-01

    Iron incorporated into food can induce precipitation and unwanted interaction with other components in food. Iron-binding proteins represent a possibility to avoid these problems and other side effects, as the iron is protected. However, there are several technical problems associated with protein–iron complex formation. In this paper, the iron-binding phosphorylated human-like collagen (Fe-G6P-HLC) was prepared under physiological conditions through phosphorylated modification. One molecule of Fe-G6P-HLC possesses about 24 atoms of Fe. Spectroscopy analysis, differential scanning calorimetry (DSC) and equilibrium dialysis techniques were employed to investigate the characteristics of the Fe-G6P-HLC. The binding sites (n b ) and apparent association constant (K app ) between iron and phosphorylated HLC were measured at n b = 23.7 and log K app = 4.57, respectively. The amount of iron (Fe 2+ sulfate) binding to phosphorylated HLC was found to be a function of pH and phosphate content. In addition, the solubility and thermal stability of HLC were not significantly affected. The results should facilitate the utilization of HLC as a bioactive iron supplement in the food and medical industry and provide an important theoretical evidence for the application of HLC chelates. - Highlights: • The iron-binding phosphorylated human-like collagen (Fe-G6P-HLC) was prepared. • One molecule of Fe-G6P-HLC possesses about 24 atoms of Fe. • The binding properties could be modulated through alterations in pH and phosphate content presented in HLC. • A novel strategy for preparing iron-binding proteins was provided

  7. Solution structure of human intestinal fatty acid binding protein: Implications for ligand entry and exit

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Fengli [Boston University School of Medicine, Department of Biophysics (United States); Luecke, Christian [Johann Wolfgang Goethe-Universitaet (Germany); Baier, Leslie J. [NIDDK, NIH, Phoenix Epidemiology and Clinical Research Branch (United States); Sacchettini, James C. [Texas A and M University, Department of Biochemistry and Biophysics (United States); Hamilton, James A. [Boston University School of Medicine, Department of Biophysics (United States)

    1997-04-15

    The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) protein which binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanine to threonine substitution at position 54 in I-FABP has been identified which affects fatty acid binding and transport, and is associated with the development of insulin resistance in several populations including Mexican-Americans and Pima Indians. To investigate the molecular basis of the binding properties of I-FABP, the 3D solution structure of the more common form of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy.Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed by using 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra(NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP was calculated by using the distance geometry program DIANA based on 2519 distance constraints obtained from the NMR data. Subsequent energy minimization was carried out by using the program SYBYL in the presence of distance constraints. The conformation of human I-FABP consists of 10 antiparallel {beta}-strands which form two nearly orthogonal {beta}-sheets of five strands each, and two short {alpha}-helices that connect the {beta}-strands A and B. The interior of the protein consists of a water-filled cavity between the two {beta}-sheets. The NMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP.The NMR results show significant conformational variability of certain backbone segments around the postulated portal region for the entry and exit of fatty acid ligand.

  8. Solution structure of human intestinal fatty acid binding protein: Implications for ligand entry and exit

    International Nuclear Information System (INIS)

    Zhang Fengli; Luecke, Christian; Baier, Leslie J.; Sacchettini, James C.; Hamilton, James A.

    1997-01-01

    The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) protein which binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanine to threonine substitution at position 54 in I-FABP has been identified which affects fatty acid binding and transport, and is associated with the development of insulin resistance in several populations including Mexican-Americans and Pima Indians. To investigate the molecular basis of the binding properties of I-FABP, the 3D solution structure of the more common form of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy.Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed by using 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra(NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP was calculated by using the distance geometry program DIANA based on 2519 distance constraints obtained from the NMR data. Subsequent energy minimization was carried out by using the program SYBYL in the presence of distance constraints. The conformation of human I-FABP consists of 10 antiparallel β-strands which form two nearly orthogonal β-sheets of five strands each, and two short α-helices that connect the β-strands A and B. The interior of the protein consists of a water-filled cavity between the two β-sheets. The NMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP.The NMR results show significant conformational variability of certain backbone segments around the postulated portal region for the entry and exit of fatty acid ligand

  9. Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

    Directory of Open Access Journals (Sweden)

    Candolfi Ermanno

    2011-07-01

    Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

  10. New human erythrocyte protein with binding sites for both spectrin and calmodulin

    International Nuclear Information System (INIS)

    Gardner, K.; Bennett, V.

    1986-01-01

    A new cytoskeletal protein that binds calmodulin has been purified to greater than 95% homogeneity from human erythrocyte cytoskeletons. The protein is a heterodimer with subunits of 103,000 and 97,000 and M/sub r/ = 197,000 calculated from its Stokes radius of 6.9 nm and sedimentation coefficient of 6.8. A binding affinity of this protein for calmodulin has been estimated to be 230 nM by displacement of two different concentrations of 125 I-azidocalmodulin with increasing concentrations of unmodified calmodulin followed by Dixon plot analysis. This protein is present in red cells at approximately 30,000 copies per cell and contains a very tight binding site(s) on cytoskeletons. The protein can be only partially solubilized from isolated cytoskeletons in buffers containing high salt, but can be totally solubilized from red cell ghost membranes by extraction in low ionic strength buffers. Affinity purified IgG against this calmodulin-binding protein identifies crossreacting polypeptide(s) in brain, kidney, testes and retina. Visualization of the calmodulin-binding protein by negative staining, rotary shadowing and unidirectional shadowing indicate that it is a flattened circular molecule with molecular height of 5.4 nm and a diameter of 12.4 nm. Preliminary cosedimentation studies with purified spectrin and F-actin indicate that the site of interaction of this calmodulin-binding protein with the cytoskeleton resides on spectrin

  11. Inhibition of platelet [3H]- imipramine binding by human plasma protein fractions

    International Nuclear Information System (INIS)

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity [ 3 H]-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha 1 acid glycoprotein, high density and low density lipoprotein, IgG and α 1 -antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of [ 3 H]-imipramine binding site

  12. Myosin phosphorylation improves contractile economy of mouse fast skeletal muscle during staircase potentiation.

    Science.gov (United States)

    Bunda, Jordan; Gittings, William; Vandenboom, Rene

    2018-01-30

    Phosphorylation of the myosin regulatory light chain (RLC) by skeletal myosin light chain kinase (skMLCK) potentiates rodent fast twitch muscle but is an ATP-requiring process. Our objective was to investigate the effect of skMLCK-catalyzed RLC phosphorylation on the energetic cost of contraction and the contractile economy (ratio of mechanical output to metabolic input) of mouse fast twitch muscle in vitro (25°C). To this end, extensor digitorum longus (EDL) muscles from wild-type (WT) and from skMLCK-devoid (skMLCK -/- ) mice were subjected to repetitive low-frequency stimulation (10 Hz for 15 s) to produce staircase potentiation of isometric twitch force, after which muscles were quick frozen for determination of high-energy phosphate consumption (HEPC). During stimulation, WT muscles displayed significant potentiation of isometric twitch force while skMLCK -/- muscles did not (i.e. 23% versus 5% change, respectively). Consistent with this, RLC phosphorylation was increased ∼3.5-fold from the unstimulated control value in WT but not in skMLCK -/- muscles. Despite these differences, the HEPC of WT muscles was not greater than that of skMLCK -/- muscles. As a result of the increased contractile output relative to HEPC, the calculated contractile economy of WT muscles was greater than that of skMLCK -/- muscles. Thus, our results suggest that skMLCK-catalyzed phosphorylation of the myosin RLC increases the contractile economy of WT mouse EDL muscle compared with skMLCK -/- muscles without RLC phosphorylation. © 2018. Published by The Company of Biologists Ltd.

  13. Clinical assessment of serum myosin light chain I in patients with dilated cardiomyopathy

    Energy Technology Data Exchange (ETDEWEB)

    Tsuda, Takashi; Izumi, Tohru; Shibata, Akira (Niigata Univ. (Japan). School of Medicine)

    1992-08-01

    Serum cardiac myosin light chain I (LCI) levels were quantitated using a radioimmunoassay kit in patients suspected of dilated cardiomyopathy (DCM). In this study, 55 patients were evaluated between 1986 and 1991. They were composed of 40 males and 15 females, and their age was 27-75 years (51[+-]11 years). The patients with renal dysfunction were excluded due to their serum creatinine levels (>2.0 mg/dl). After cardiac catheterization, endomyocardial biopsy and echocardiography, 44 patients were diagnosed as DCM, 2 as ischemic heart disease, 2 as chronic myocarditis, 1 as restrictive cardiomyopathy, 1 as dilated hypertrophic cardiomyopathy, 1 as cardiac amyloidosis, 2 as myopathy, 1 as polymyositis and 1 as hypothyroidism. Only two patients with DCM had elevated LCI. Besides, two patients with myopathy or hypothyroidism had elevated LCI. In the follow-up, one patient died suddenly 6 months later and another showed normal value of LCI four years later. LCI elevation in DCM was not related to either the severity of heart failure or cardiac function and it showed no finding of [sup 201]Tl myocardial defect or elevated CPK. The mechanism for elevated LCI in myopathy is related to a crossreaction with myosin light chain in the skeletal muscle. In hypothyroidism, it may be related to decreased clearance of normal LCI concentration or increased myosin light chain from damaged skeletal muscle. In conclusion, it is evident that the measurement of LCI is not helpful in clinical assessment of patients with DCM, but may be useful in detection of secondary cardiomyopathy. (author).

  14. Clinical assessment of serum myosin light chain I in patients with dilated cardiomyopathy

    International Nuclear Information System (INIS)

    Tsuda, Takashi; Izumi, Tohru; Shibata, Akira

    1992-01-01

    Serum cardiac myosin light chain I (LCI) levels were quantitated using a radioimmunoassay kit in patients suspected of dilated cardiomyopathy (DCM). In this study, 55 patients were evaluated between 1986 and 1991. They were composed of 40 males and 15 females, and their age was 27-75 years (51±11 years). The patients with renal dysfunction were excluded due to their serum creatinine levels (>2.0 mg/dl). After cardiac catheterization, endomyocardial biopsy and echocardiography, 44 patients were diagnosed as DCM, 2 as ischemic heart disease, 2 as chronic myocarditis, 1 as restrictive cardiomyopathy, 1 as dilated hypertrophic cardiomyopathy, 1 as cardiac amyloidosis, 2 as myopathy, 1 as polymyositis and 1 as hypothyroidism. Only two patients with DCM had elevated LCI. Besides, two patients with myopathy or hypothyroidism had elevated LCI. In the follow-up, one patient died suddenly 6 months later and another showed normal value of LCI four years later. LCI elevation in DCM was not related to either the severity of heart failure or cardiac function and it showed no finding of 201 Tl myocardial defect or elevated CPK. The mechanism for elevated LCI in myopathy is related to a crossreaction with myosin light chain in the skeletal muscle. In hypothyroidism, it may be related to decreased clearance of normal LCI concentration or increased myosin light chain from damaged skeletal muscle. In conclusion, it is evident that the measurement of LCI is not helpful in clinical assessment of patients with DCM, but may be useful in detection of secondary cardiomyopathy. (author)

  15. Binding of recombinant HIV coat protein gp120 to human monocytes

    International Nuclear Information System (INIS)

    Finbloom, D.S.; Hoover, D.L.; Meltzer, M.S.

    1991-01-01

    Inasmuch as the exact level of CD4 Ag expression on macrophages is controversial and because HIV may interact with macrophages in a manner different from that on T cells, we analyzed the binding of gp120 to freshly isolated and cultured monocytes. rgp120 was iodinated using the lactoperoxidase method to a sp. act. of 600 Ci/mmol. Highly purified monocytes (greater than 90%) were isolated from the leukapheresed blood of normal volunteers by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation and cultured 7 days in DMEM supplemented with 1000 U/ml macrophage CSF in 10% human serum. Whereas MOLT/4 cells consistently bound freshly prepared 125I-rgp120 at 80% specificity with 5100 +/- 700 mol/cell, MCSF cultured monocytes bound rgp120 at only 0 to 20% specificity and 420 +/- 200 mol/cell. Most of the radioactivity bound by these cells could not be blocked by the addition of unlabeled rgp120. In contrast, the U937 myeloid cell line bound rgp120 with 50% specificity and about 2500 mol/cell. Whereas the antibody OKT4a (anti-CD4) blocked 80% of the binding on MOLT/4 cells and 50% on U937 cells, binding was only inhibited on the average of 6% on cultured monocytes. When soluble rCD4 was used as an inhibitor, binding to MOLT/4 cells was blocked by 80%. In contrast, binding to cultured monocytes was inhibited by 28%. HIV infectivity was blocked by similar concentrations of OKT4a. These observations suggest that although most binding of gp120 to cultured monocytes is not to the CD4 determinant, several hundred molecules do bind to a CD4-like molecule which promotes virus entry and replication

  16. The importance of subfragment 2 and C-terminus of myosin heavy chain for thick filament assembly in skeletal muscle cells.

    Science.gov (United States)

    Ojima, Koichi; Oe, Mika; Nakajima, Ikuyo; Shibata, Masahiro; Muroya, Susumu; Chikuni, Koichi; Hattori, Akihito; Nishimura, Takanori

    2015-04-01

    In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 (S2) + light meromyosin (LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C-terminus on thick filament assembly. C-terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C-terminus of LMM. © 2014 Japanese Society of Animal Science.

  17. Molecular docking and NMR binding studies to identify novel inhibitors of human phosphomevalonate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Boonsri, Pornthip [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States); Department of Chemistry, NANOTEC Center of Nanotechnology, National Nanotechnology Center, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Neumann, Terrence S.; Olson, Andrew L.; Cai, Sheng [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States); Herdendorf, Timothy J.; Miziorko, Henry M. [Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110 (United States); Hannongbua, Supa [Department of Chemistry, NANOTEC Center of Nanotechnology, National Nanotechnology Center, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Sem, Daniel S., E-mail: daniel.sem@cuw.edu [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Natural and synthetic inhibitors of human phosphomevalonate kinase identified. Black-Right-Pointing-Pointer Virtual screening yielded a hit rate of 15%, with inhibitor K{sub d}'s of 10-60 {mu}M. Black-Right-Pointing-Pointer NMR studies indicate significant protein conformational changes upon binding. -- Abstract: Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using autodock. Promising hits were verified and their affinity measured using NMR-based {sup 1}H-{sup 15}N heteronuclear single quantum coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (K{sub d}). Tight binding compounds with K{sub d}'s ranging from 6-60 {mu}M were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC cross peak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development.

  18. The binding patterns of antisera to sex steroids and human gonadotropins on human and rhesus monkey spermatozoa.

    Science.gov (United States)

    Allag, I S; Das, R P; Roy, S

    1983-01-01

    The presence of different hormones on the surface of ejaculated spermatozoa was determined by immunofluorescence studies of the binding patterns of specific antisera to these hormones. There were striking similarities in the binding pattern of antisera to steroid hormones found on human and monkey spermatozoa. Assuming the intensity of fluorescence is proportional to the concentration of the hormone, concentrations of testosterone on the acrosomal and the postacrosomal regions were higher than levels of progesterone and estrogens. Spermatozoa with a "tapering head" had more hCG bound on the acrosomal and postacrosomal regions than spermatozoa with "normal head" (oval shaped). Correlating these findings to the functions of spermatozoa will require further studies.

  19. Construction and characterization of the alpha form of a cardiac myosin heavy chain cDNA clone and its developmental expression in the Syrian hamster.

    OpenAIRE

    Liew, C C; Jandreski, M A

    1986-01-01

    A cDNA clone, pVHC1, was isolated from a Syrian hamster heart cDNA library and was compared to the rat alpha (pCMHC21) and beta (pCMHC5) ventricular myosin heavy chain cDNA clones. The DNA sequence and amino acid sequence deducted from the DNA show more homology with pCMHC21 than pCMHC5. This indicates that pVHC1 is an alpha ventricular myosin heavy chain cDNA clone. However, even though pVHC1 shows a high degree of nucleotide and amino acid conservation with the rat myosin heavy chain sequen...

  20. Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function

    Science.gov (United States)

    Vandenboom, Rene; Herron, Todd; Favre, Elizabeth; Albayya, Faris P.

    2011-01-01

    The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a “headless” myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC + endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P < 0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules. PMID:21112946

  1. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

    Energy Technology Data Exchange (ETDEWEB)

    Lima, V.V. [Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso, Barra do Garças, MT (Brazil); Lobato, N.S.; Filgueira, F.P. [Curso de Medicina, Setor de Fisiologia Humana, Universidade Federal de Goiás, Jataí, GO (Brazil); Webb, R.C. [Department of Physiology, Georgia Regents University, Augusta, GA (United States); Tostes, R.C. [Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Giachini, F.R. [Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso, Barra do Garças, MT (Brazil)

    2014-08-15

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca{sup 2+}/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

  2. Nuclear myosin is ubiquitously expressed and evolutionary conserved in vertebrates

    Czech Academy of Sciences Publication Activity Database

    Kahle, Michal; Přidalová, Jarmila; Špaček, M.; Dzijak, Rastislav; Hozák, Pavel

    2007-01-01

    Roč. 127, č. 2 (2007), s. 139-184 ISSN 0948-6143 R&D Projects: GA ČR GA204/04/0108; GA AV ČR IAA5039202; GA MŠk LC545; GA ČR GD204/05/H023 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50520514 Keywords : Nuclear myosin I * Transcription * Chromatin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.893, year: 2007

  3. Preparation of biologically active 32P-labeled human relaxin. Displaceable binding to rat uterus, cervix, and brain

    International Nuclear Information System (INIS)

    Osheroff, P.L.; Ling, V.T.; Vandlen, R.L.; Cronin, M.J.; Lofgren, J.A.

    1990-01-01

    Relaxin is a member of the insulin family of polypeptide hormones and is known to exert its biological effects on various parts of the mammalian reproductive system. Biologically active human relaxin has been chemically synthesized based on the nucleotide sequence obtained from an ovarian cDNA clone. In the present study synthetic human relaxin was radiolabled by phosphorylation with cAMP-dependent protein kinase and [gamma-32P]ATP to a specific activity of 5000 Ci/mmol. The phosphorylated relaxin was purified on cation exchange high performance liquid chromatography and was shown to co-migrate with relaxin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed a single phosphorylated site on the B chain of relaxin. The 32P-relaxin was able to bind to a goat anti-relaxin antibody, and this binding could be displaced by unlabeled relaxin in a concentration-dependent manner. A comparison of the concentration responses of cellular cAMP production stimulated by relaxin and phosphorylated relaxin in a primary human uterine cell line showed that phosphorylation did not affect the in vitro biological efficacy of relaxin. This made it suitable for in situ autoradiographic localization of relaxin binding sites in rat uterine, cervical, and brain tissue sections. Displacement of the binding of 100 pM 32P-relaxin by 100, 10, and 3 nM unlabeled relaxin, but not by 100 nM insulin, insulin-like growth factor-I, and an insulin-like growth factor-I analog, demonstrated the high affinity and specificity of such binding. We conclude that 32P-labeled human relaxin is biologically and immunologically active and that this novel probe binds reversibly and with high affinity to classical (e.g. uterus) and unpredicted (e.g. brain) tissues

  4. Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography

    International Nuclear Information System (INIS)

    Gillberg, P.-G.; Aquilonius, S.-M.

    1985-01-01

    Binding sites for the receptor ligands 3 H-quinuclidinylbenzilate, 3 H-alpha-bungarotoxin ( 3 H-alpha-Btx), 3 H-etorphine and 3 H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3 H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distribute within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged. (author)

  5. Binding of digitoxin and digoxin to normal human β-lipoproteins

    International Nuclear Information System (INIS)

    Brock, A.

    1976-01-01

    The binding of digitoxin and digoxin to purified β-lipoprotein, obtained from pooled normal human serum, was studied under equilibrium conditions. Even with as high concentrations of unbound digitoxin or digoxin as 4 μmol/l, the preparations of β-lipoproteins, containing cholesterol 1.98-3.95 mmol/l, showed no signs of saturation. The binding affinity of digitoxin was about ten times as high as that of digoxin. Gel filtration chromatography, performed on native serum after addition of 3 H-digitoxin or 3 H-digoxin, showed a minor fraction of the cardiac glycosides to be associated with te protein fraction of highest molecular weight. This phenomenon disappeared after precipitation of the β-lipoproteins. In clinical relations the contribution of protein-bound digitoxin caused by the lipoprotein interaction is immaterial compared to that caused by the albumin interaction. (author)

  6. Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

    Science.gov (United States)

    Tabernero, L; Chang, C Y; Ohringer, S L; Lau, W F; Iwanowicz, E J; Han, W C; Wang, T C; Seiler, S M; Roberts, D G; Sack, J S

    1995-02-10

    The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

  7. Chromosomal localization of the human diazepam binding inhibitor gene

    International Nuclear Information System (INIS)

    DeBernardi, M.A.; Crowe, R.R.; Mocchetti, I.; Shows, T.B.; Eddy, R.L.; Costa, E.

    1988-01-01

    The authors have used in situ chromosome hybridization and human-mouse somatic cell hybrids to map the gene(s) for human diazepam binding inhibitor (DBI), an endogenous putative modulator of the γ-aminobutyric acid receptor acting at the allosteric regulatory center of this receptor that includes the benzodiazepine recognition site. In 784 chromosome spreads hybridized with human DBI cDNA, the distribution of 1,476 labeled sites revealed a significant clustering of autoradiographic grains (11.3% of total label) on the long arm of chromosome 2 (2q). Furthermore, 63.5% of the grains found on 2q were located on 2q12-21, suggesting regional mapping of DBI gene(s) to this segment. Secondary hybridization signals were frequently observed on other chromosomes and they were statistically significant mainly for chromosomes 5, 6, 11, and 14. In addition, DNA from 32 human-mouse cell hybrids was digested with BamHI and probed with human DBI cDNA. A 3.5-kilobase band, which probably represents the human DBI gene, was assigned to chromosome 2. Four higher molecular weight bands, also detected in BamHI digests, could not be unequivocally assigned. A chromosome 2 location was excluded for the 27-, 13-, and 10-kilobase bands. These results assign a human DBI gene to chromosome 2 (2q12-21) and indicate that three of the four homologous sequences detected by the human DBI probe are located on three other chromosomes

  8. Human TFDP3, a novel DP protein, inhibits DNA binding and transactivation by E2F

    DEFF Research Database (Denmark)

    Qiao, Huan; Di Stefano, Luisa; Tian, Chan

    2006-01-01

    The two known DP proteins, TFDP1 and -2, bind E2Fs to form heterodimers essential for high affinity DNA binding and efficient transcriptional activation/repression. Here we report the identification of a new member of the DP family, human TFDP3. Despite the high degree of sequence similarity, TFD...

  9. [3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts

    International Nuclear Information System (INIS)

    Shoemaker, D.G.; Lauf, P.K.

    1983-01-01

    The interaction of the cardiac glycoside [ 3 H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [ 3 H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [ 3 H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [ 3 H]ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor

  10. Interaction of complement-solubilized immune complexes with CR1 receptors on human erythrocytes. The binding reaction

    DEFF Research Database (Denmark)

    Jepsen, H H; Svehag, S E; Jarlbaek, L

    1986-01-01

    showed no binding. IC solubilized in 50% human serum in the presence of autologous RBC bound rapidly to RBC-CR1, with maximal binding within less than 1 min at 37 degrees C. Release of CR1-bound IC under these conditions occurred slowly, requiring more than 30 min. Only binding of 'partially' solubilized...... of an intact classical pathway in preparing the IC for binding to RBC-CR1. C-solubilized IC could be absorbed to solid-phase conglutinin or antibody to C3c and C4c, and these ligands were able to inhibit the binding of solubilized IC to RBC. Heparin also exerted a marked, dose-dependent inhibitory effect...

  11. Theta Phase Synchronization Is the Glue that Binds Human Associative Memory.

    Science.gov (United States)

    Clouter, Andrew; Shapiro, Kimron L; Hanslmayr, Simon

    2017-10-23

    Episodic memories are information-rich, often multisensory events that rely on binding different elements [1]. The elements that will constitute a memory episode are processed in specialized but distinct brain modules. The binding of these elements is most likely mediated by fast-acting long-term potentiation (LTP), which relies on the precise timing of neural activity [2]. Theta oscillations in the hippocampus orchestrate such timing as demonstrated by animal studies in vitro [3, 4] and in vivo [5, 6], suggesting a causal role of theta activity for the formation of complex memory episodes, but direct evidence from humans is missing. Here, we show that human episodic memory formation depends on phase synchrony between different sensory cortices at the theta frequency. By modulating the luminance of visual stimuli and the amplitude of auditory stimuli, we directly manipulated the degree of phase synchrony between visual and auditory cortices. Memory for sound-movie associations was significantly better when the stimuli were presented in phase compared to out of phase. This effect was specific to theta (4 Hz) and did not occur in slower (1.7 Hz) or faster (10.5 Hz) frequencies. These findings provide the first direct evidence that episodic memory formation in humans relies on a theta-specific synchronization mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Autoradiographic quantification of vasoactive intestinal peptide binding sites in sections from human blood mononuclear cell pellets

    Energy Technology Data Exchange (ETDEWEB)

    Gutkind, J.S.; Kurihara, M.; Castren, E.; Saavedra, J.M.

    1988-09-01

    Quantitative autoradiographic methods were utilized to characterize specific, high-affinity vasoactive intestinal peptide binding sites (Kd = 310 +/- 60 pmol/L; Bmax = 93 +/- 11 fmol/mg protein) in frozen sections obtained from a mononuclear cell pellet derived from 20 ml of human blood. The method is at least one order of magnitude more sensitive than conventional membrane binding techniques, and it has the potential for wide applications in studies of neuropeptide, biogenic amine, and drug binding in clinical samples.

  13. Autoradiographic quantification of vasoactive intestinal peptide binding sites in sections from human blood mononuclear cell pellets

    International Nuclear Information System (INIS)

    Gutkind, J.S.; Kurihara, M.; Castren, E.; Saavedra, J.M.

    1988-01-01

    Quantitative autoradiographic methods were utilized to characterize specific, high-affinity vasoactive intestinal peptide binding sites (Kd = 310 +/- 60 pmol/L; Bmax = 93 +/- 11 fmol/mg protein) in frozen sections obtained from a mononuclear cell pellet derived from 20 ml of human blood. The method is at least one order of magnitude more sensitive than conventional membrane binding techniques, and it has the potential for wide applications in studies of neuropeptide, biogenic amine, and drug binding in clinical samples

  14. A novel de novo mutation of β-cardiac myosin heavy chain gene ...

    Indian Academy of Sciences (India)

    2014-08-18

    Aug 18, 2014 ... It is one of the most important diseases causing a sud- den death in ... familial HCM encode contractile proteins such as β-cardiac myosin ... A previously healthy 12-year-old boy was admitted to our hospital for .... Maron B. J. 2002 Hypertrophic cardiomyopathy: A systematic review. JAMA 287, 1308–1320.

  15. Formation and characterization of iron-binding phosphorylated human-like collagen as a potential iron supplement

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Jianjun; Chen, Fei; Fan, Daidi, E-mail: fandaidi@nwu.edu.cn; Zhu, Chenhui; Ma, Xiaoxuan; Xue, Wenjiao

    2013-10-01

    Iron incorporated into food can induce precipitation and unwanted interaction with other components in food. Iron-binding proteins represent a possibility to avoid these problems and other side effects, as the iron is protected. However, there are several technical problems associated with protein–iron complex formation. In this paper, the iron-binding phosphorylated human-like collagen (Fe-G6P-HLC) was prepared under physiological conditions through phosphorylated modification. One molecule of Fe-G6P-HLC possesses about 24 atoms of Fe. Spectroscopy analysis, differential scanning calorimetry (DSC) and equilibrium dialysis techniques were employed to investigate the characteristics of the Fe-G6P-HLC. The binding sites (n{sub b}) and apparent association constant (K{sub app}) between iron and phosphorylated HLC were measured at n{sub b} = 23.7 and log K{sub app} = 4.57, respectively. The amount of iron (Fe{sup 2+} sulfate) binding to phosphorylated HLC was found to be a function of pH and phosphate content. In addition, the solubility and thermal stability of HLC were not significantly affected. The results should facilitate the utilization of HLC as a bioactive iron supplement in the food and medical industry and provide an important theoretical evidence for the application of HLC chelates. - Highlights: • The iron-binding phosphorylated human-like collagen (Fe-G6P-HLC) was prepared. • One molecule of Fe-G6P-HLC possesses about 24 atoms of Fe. • The binding properties could be modulated through alterations in pH and phosphate content presented in HLC. • A novel strategy for preparing iron-binding proteins was provided.

  16. Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Kailang; Li, Weikai; Peng, Guiqing; Li, Fang; (Harvard-Med); (UMM-MED)

    2010-03-04

    NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel {beta}-sandwich core structure consisting of 2 layers of {beta}-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homology in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a 'virus-binding hotspot' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.

  17. Bat Caliciviruses and Human Noroviruses Are Antigenically Similar and Have Overlapping Histo-Blood Group Antigen Binding Profiles.

    Science.gov (United States)

    Kocher, Jacob F; Lindesmith, Lisa C; Debbink, Kari; Beall, Anne; Mallory, Michael L; Yount, Boyd L; Graham, Rachel L; Huynh, Jeremy; Gates, J Edward; Donaldson, Eric F; Baric, Ralph S

    2018-05-22

    Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens. IMPORTANCE Caliciviruses are

  18. STATISTICAL INSIGHT INTO THE BINDING REGIONS IN DISORDERED HUMAN PROTEOME

    Directory of Open Access Journals (Sweden)

    Uttam Pal

    2016-03-01

    Full Text Available The human proteome contains a significant number of intrinsically disordered proteins (IDPs. They show unusual structural features that enable them to participate in diverse cellular functions and play significant roles in cell signaling and reorganization processes. In addition, the actions of IDPs, their functional cooperativity, conformational alterations and folding often accompany binding to a target macromolecule. Applying bioinformatics approaches and with the aid of statistical methodologies, we investigated the statistical parameters of binding regions (BRs found in disordered human proteome. In this report, we detailed the bioinformatics analysis of binding regions found in the IDPs. Statistical models for the occurrence of BRs, their length distribution and percent occupancy in the parent proteins are shown. The frequency of BRs followed a Poisson distribution pattern with increasing expectancy with the degree of disorderedness. The length of the individual BRs also followed Poisson distribution with a mean of 6 residues, whereas, percentage of residues in BR showed a normal distribution pattern. We also explored the physicochemical properties such as the grand average of hydropathy (GRAVY and the theoretical isoelectric points (pIs. The theoretical pIs of the BRs followed a bimodal distribution as in the parent proteins. However, the mean acidic/basic pIs were significantly lower/higher than that of the proteins, respectively. We further showed that the amino acid composition of BRs was enriched in hydrophobic residues such as Ala, Val, Ile, Leu and Phe compared to the average sequence content of the proteins. Sequences in a BR showed conformational adaptability mostly towards flexible coil structure and followed by helix, however, the ordered secondary structural conformation was significantly lower in BRs than the proteins. Combining and comparing these statistical information of BRs with other methods may be useful for high

  19. Cyanide binding to human plasma heme–hemopexin: A comparative study

    International Nuclear Information System (INIS)

    Ascenzi, Paolo; Leboffe, Loris; Polticelli, Fabio

    2012-01-01

    Highlights: ► Cyanide binding to ferric HHPX–heme–Fe. ► Cyanide binding to ferrous HHPX–heme–Fe. ► Dithionite-mediated reduction of ferric HHPX–heme–Fe–cyanide. ► Cyanide binding to HHPX–heme–Fe is limited by ligand deprotonation. ► Cyanide dissociation from HHPX–heme–Fe–cyanide is limited by ligand protonation. -- Abstract: Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme–Fe–hemopexin (HPX–heme–Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX–heme–Fe (HHPX–heme–Fe(III) and HHPX–heme–Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX–heme–Fe(III)–cyanide complex, at pH 7.4 and 20.0 °C, are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX–heme–Fe(III) and HHPX–heme–Fe(II) are K = (4.1 ± 0.4) × 10 −6 M, k on = (6.9 ± 0.5) × 10 1 M −1 s −1 , and k off = 2.8 × 10 −4 s −1 ; and H = (6 ± 1) × 10 −1 M, h on = 1.2 × 10 −1 M −1 s −1 , and h off = (7.1 ± 0.8) × 10 −2 s −1 , respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX–heme–Fe(III)–cyanide complex is l = 8.9 ± 0.8 M −1/2 s −1 . HHPX–heme–Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively.

  20. Identification and molecular modelling of a mutation in the motor head domain of myosin VIIA in a family with autosomal dominant hearing impairment (DFNA11)

    NARCIS (Netherlands)

    Luijendijk, M.W.J.; Wijk, E. van; Bischoff, A.M.L.C.; Krieger, E.; Huygen, P.L.M.; Pennings, R.J.E.; Brunner, H.G.; Cremers, C.W.R.J.; Cremers, F.P.M.; Kremer, J.M.J.

    2004-01-01

    Myosin VIIA is an unconventional myosin that has been implicated in Usher syndrome type 1B, atypical Usher syndrome, non-syndromic autosomal recessive hearing impairment (DFNB2) and autosomal dominant hearing impairment (DFNA11). Here, we present a family with non-syndromic autosomal dominant

  1. Identification and molecular modelling of a mutation in the motor head domain of myosin VIIA in a family with autosomal dominant hearing impairment (DFNA11).

    NARCIS (Netherlands)

    Luijendijk, M.W.J.; Wijk, E. van; Bischoff, A.M.L.C.; Krieger, E.; Huygen, P.L.M.; Pennings, R.J.E.; Brunner, H.G.; Cremers, C.W.R.J.; Cremers, F.P.M.; Kremer, J.M.J.

    2004-01-01

    Myosin VIIA is an unconventional myosin that has been implicated in Usher syndrome type 1B, atypical Usher syndrome, non-syndromic autosomal recessive hearing impairment (DFNB2) and autosomal dominant hearing impairment (DFNA11). Here, we present a family with non-syndromic autosomal dominant

  2. Maintenance of muscle myosin levels in adult C. elegans requires both the double bromodomain protein BET-1 and sumoylation

    Directory of Open Access Journals (Sweden)

    Kate Fisher

    2013-10-01

    Attenuation of RAS-mediated signalling is a conserved process essential to control cell proliferation, differentiation, and apoptosis. Cooperative interactions between histone modifications such as acetylation, methylation and sumoylation are crucial for proper attenuation in C. elegans, implying that the proteins recognising these histone modifications could also play an important role in attenuation of RAS-mediated signalling. We sought to systematically identify these proteins and found BET-1. BET-1 is a conserved double bromodomain protein that recognises acetyl-lysines on histone tails and maintains the stable fate of various lineages. Unexpectedly, adults lacking both BET-1 and SUMO-1 are depleted of muscle myosin, an essential component of myofibrils. We also show that this muscle myosin depletion does not occur in all animals at a specific time, but rather that the penetrance of the phenotype increases with age. To gain mechanistic insights into this process, we sought to delay the occurrence of the muscle myosin depletion phenotype and found that it requires caspase activity and MEK-dependent signalling. We also performed transcription profiling on these mutants and found an up-regulation of the FGF receptor, egl-15, a tyrosine kinase receptor acting upstream of MEK. Consistent with a MEK requirement, we could delay the muscle phenotype by systemic or hypodermal knock down of egl-15. Thus, this work uncovered a caspase- and MEK-dependent mechanism that acts specifically on ageing adults to maintain the appropriate net level of muscle myosin.

  3. Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

    Science.gov (United States)

    Sriram, M; Osipiuk, J; Freeman, B; Morimoto, R; Joachimiak, A

    1997-03-15

    The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes

  4. 125I-human epidermal growth factor specific binding to placentas and fetal membranes from varoius pregnancy states

    International Nuclear Information System (INIS)

    Hofmann, G.E.; Siddiqi, T.A.; Rao, Ch. V.; Carman, F.R.

    1988-01-01

    Specific binding of 125 I-human epidermal growth factor (hEGF) to homogenates of term human placentas and fetal membranes from normal and appropriate for gestational age (N = 20), intrauterine growth retarded (N = 9), twin (N = 11), White class A/B diabetic (N = 12), and large for gestational age (N = 13) pregnancies was measured. In all pregnancy states, placentas bound approximately four times more 125 I-hEGF than did fetal membranes (P 125 I-hEGF binding to fetal membranes from the various pregnancy states (P 125 I-hEGF specific binding to placentas from intrauterine growth retarded or twin pregnancies was significantly greater compared with placentas from normal and appropriate for gestational age pregnancies (P 125 I-hEGF specific binding did not differ between placentas from intrauterine growth retarded or twin pregnancies (P 125 I-hEGF binding did not vary with fetal sex, maternal race, placental weight, or gestational age between 37 to 42 weeks (P 125 I-hEGF binding increased with increasing infant weight when appropriate for gestational age and large for gestational age infants were included (P<0.05, r = 0.38, N = 32) but not for intrauterine growth retarded, appropriate for gestational age, or large for gestational age infants alone. (author)

  5. Two distinct affinity binding sites for IL-1 on human cell lines

    International Nuclear Information System (INIS)

    Bensimon, C.; Wakasugi, N.; Tagaya, Y.; Takakura, K.; Yodoi, J.; Tursz, T.; Wakasugi, H.

    1989-01-01

    We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125 I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated

  6. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

    DEFF Research Database (Denmark)

    Green, M R; Couchman, J R

    1985-01-01

    , the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing......Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map...... whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar...

  7. Delineation of the peptide binding site of the human galanin receptor.

    Science.gov (United States)

    Kask, K; Berthold, M; Kahl, U; Nordvall, G; Bartfai, T

    1996-01-01

    Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th Images PMID:8617199

  8. Protein multi-scale organization through graph partitioning and robustness analysis: application to the myosin–myosin light chain interaction

    International Nuclear Information System (INIS)

    Delmotte, A; Barahona, M; Tate, E W; Yaliraki, S N

    2011-01-01

    Despite the recognized importance of the multi-scale spatio-temporal organization of proteins, most computational tools can only access a limited spectrum of time and spatial scales, thereby ignoring the effects on protein behavior of the intricate coupling between the different scales. Starting from a physico-chemical atomistic network of interactions that encodes the structure of the protein, we introduce a methodology based on multi-scale graph partitioning that can uncover partitions and levels of organization of proteins that span the whole range of scales, revealing biological features occurring at different levels of organization and tracking their effect across scales. Additionally, we introduce a measure of robustness to quantify the relevance of the partitions through the generation of biochemically-motivated surrogate random graph models. We apply the method to four distinct conformations of myosin tail interacting protein, a protein from the molecular motor of the malaria parasite, and study properties that have been experimentally addressed such as the closing mechanism, the presence of conserved clusters, and the identification through computational mutational analysis of key residues for binding

  9. Low nucleosome occupancy is encoded around functional human transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Daenen Floris

    2008-07-01

    Full Text Available Abstract Background Transcriptional regulation of genes in eukaryotes is achieved by the interactions of multiple transcription factors with arrays of transcription factor binding sites (TFBSs on DNA and with each other. Identification of these TFBSs is an essential step in our understanding of gene regulatory networks, but computational prediction of TFBSs with either consensus or commonly used stochastic models such as Position-Specific Scoring Matrices (PSSMs results in an unacceptably high number of hits consisting of a few true functional binding sites and numerous false non-functional binding sites. This is due to the inability of the models to incorporate higher order properties of sequences including sequences surrounding TFBSs and influencing the positioning of nucleosomes and/or the interactions that might occur between transcription factors. Results Significant improvement can be expected through the development of a new framework for the modeling and prediction of TFBSs that considers explicitly these higher order sequence properties. It would be particularly interesting to include in the new modeling framework the information present in the nucleosome positioning sequences (NPSs surrounding TFBSs, as it can be hypothesized that genomes use this information to encode the formation of stable nucleosomes over non-functional sites, while functional sites have a more open chromatin configuration. In this report we evaluate the usefulness of the latter feature by comparing the nucleosome occupancy probabilities around experimentally verified human TFBSs with the nucleosome occupancy probabilities around false positive TFBSs and in random sequences. Conclusion We present evidence that nucleosome occupancy is remarkably lower around true functional human TFBSs as compared to non-functional human TFBSs, which supports the use of this feature to improve current TFBS prediction approaches in higher eukaryotes.

  10. Human muscle proteins: analysis by two-dimensional electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Giometti, C.S.; Danon, M.J.; Anderson, N.G.

    1983-09-01

    Proteins from single frozen sections of human muscle were separated by two-dimensional gel electrophoresis and detected by fluorography or Coomassie Blue staining. The major proteins were identical in different normal muscles obtained from either sex at different ages, and in Duchenne and myotonic dystrophy samples. Congenital myopathy denervation atrophy, polymyositis, and Becker's muscular dystrophy samples, however, showed abnormal myosin light chain compositions, some with a decrease of fast-fiber myosin light chains and others with a decrease of slow-fiber light chains. These protein alterations did not correlate with any specific disease, and may be cause by generalized muscle-fiber damage.

  11. Elucidation of the binding mechanism of coumarin derivatives with human serum albumin.

    Directory of Open Access Journals (Sweden)

    Archit Garg

    Full Text Available Coumarin is a benzopyrone which is widely used as an anti-coagulant, anti-oxidant, anti-cancer and also to cure arthritis, herpes, asthma and inflammation. Here, we studied the binding of synthesized coumarin derivatives with human serum albumin (HSA at physiological pH 7.2 by using fluorescence spectroscopy, circular dichroism spectroscopy, molecular docking and molecular dynamics simulation studies. By addition of coumarin derivatives to HSA the maximum fluorescence intensity was reduced due to quenching of intrinsic fluorescence upon binding of coumarin derivatives to HSA. The binding constant and free energy were found to be 1.957±0.01×10(5 M(-1, -7.175 Kcal M(-1 for coumarin derivative (CD enamide; 0.837±0.01×10(5 M(-1, -6.685 Kcal M(-1 for coumarin derivative (CD enoate, and 0.606±0.01×10(5 M(-1, -6.49 Kcal M(-1 for coumarin derivative methylprop (CDM enamide. The CD spectroscopy showed that the protein secondary structure was partially unfolded upon binding of coumarin derivatives. Further, the molecular docking studies showed that coumarin derivatives were binding to HSA at sub-domain IB with the hydrophobic interactions and also with hydrogen bond interactions. Additionally, the molecular dynamics simulations studies contributed in understanding the stability of protein-drug complex system in the aqueous solution and the conformational changes in HSA upon binding of coumarin derivatives. This study will provide insights into designing of the new inspired coumarin derivatives as therapeutic agents against many life threatening diseases.

  12. The local expression of adult chicken heart myosins during development. II. Ventricular conducting tissue

    NARCIS (Netherlands)

    Sanders, E.; de Groot, I. J.; Geerts, W. J.; de Jong, F.; van Horssen, A. A.; Los, J. A.; Moorman, A. F.

    1986-01-01

    The development of the ventricular conducting tissue of the embryonic chicken heart has been studied using a previous finding that morphologically recognizable atrial conducting tissue coexpresses the atrial and the ventricular myosin isoforms. It is found that, by these criteria, at 9 days part of

  13. Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

    International Nuclear Information System (INIS)

    Sluss, P.M.; Krystek, S.R. Jr.; Andersen, T.T.; Melson, B.E.; Huston, J.S.; Ridge, R.; Reichert, L.E. Jr.

    1986-01-01

    Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125 I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125 I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125 I-labeled human FSH to testis receptor

  14. Fluorometric and molecular docking investigation on the binding characteristics of SB202190 to human serum albumin

    International Nuclear Information System (INIS)

    Nasruddin, Ahmad N.; Feroz, Shevin R.; Mukarram, Abdul K.; Mohamad, Saharuddin B.; Tayyab, Saad

    2016-01-01

    The interaction of SB202190, a p38 mitogen-activated protein kinase inhibitor with the main drug transporter in human circulation, human serum albumin (HSA) was studied using fluorescence spectroscopy and in silico docking methods. The association constant, K a of the binding reaction was determined to be 3.24±0.07×10 4 M −1 at 25 °C based on fluorescence quenching titration results. The values of enthalpy change and entropy change for the interaction were found as −8.54 kJ mol −1 and 58.01 J mol −1 K −1 , respectively. Both thermodynamic data and docking results suggested the involvement of hydrophobic and van der Waals forces in the complex formation. Three-dimensional fluorescence data of SB202190–HSA complex demonstrated significant changes in the microenvironment around the protein fluorophores upon drug binding. Comparison of HSA thermograms obtained in the absence and the presence of SB202190 suggested improved protein thermal stability upon complexation with the drug. Competitive drug displacement results as well as modeling data concluded the preferred binding site of SB202190 on HSA as Sudlow's site I. - Highlights: • SB202190 interacts with HSA with moderate affinity. • Involvement of hydrophobic and van der Waals forces in SB202190 binding. • SB202190 binding results in microenvironmental changes around fluorophores. • Sudlow's site I is the preferred binding site of SB202190.

  15. Fluorometric and molecular docking investigation on the binding characteristics of SB202190 to human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Nasruddin, Ahmad N.; Feroz, Shevin R. [Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Mukarram, Abdul K. [Bioinformatics Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Mohamad, Saharuddin B. [Bioinformatics Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Centre of Research for Computational Sciences and Informatics for Biology, Bioindustry, Environment, Agriculture and Healthcare, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Tayyab, Saad, E-mail: saadtayyab2004@yahoo.com [Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Centre of Research for Computational Sciences and Informatics for Biology, Bioindustry, Environment, Agriculture and Healthcare, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia)

    2016-06-15

    The interaction of SB202190, a p38 mitogen-activated protein kinase inhibitor with the main drug transporter in human circulation, human serum albumin (HSA) was studied using fluorescence spectroscopy and in silico docking methods. The association constant, K{sub a} of the binding reaction was determined to be 3.24±0.07×10{sup 4} M{sup −1} at 25 °C based on fluorescence quenching titration results. The values of enthalpy change and entropy change for the interaction were found as −8.54 kJ mol{sup −1} and 58.01 J mol{sup −1} K{sup −1}, respectively. Both thermodynamic data and docking results suggested the involvement of hydrophobic and van der Waals forces in the complex formation. Three-dimensional fluorescence data of SB202190–HSA complex demonstrated significant changes in the microenvironment around the protein fluorophores upon drug binding. Comparison of HSA thermograms obtained in the absence and the presence of SB202190 suggested improved protein thermal stability upon complexation with the drug. Competitive drug displacement results as well as modeling data concluded the preferred binding site of SB202190 on HSA as Sudlow's site I. - Highlights: • SB202190 interacts with HSA with moderate affinity. • Involvement of hydrophobic and van der Waals forces in SB202190 binding. • SB202190 binding results in microenvironmental changes around fluorophores. • Sudlow's site I is the preferred binding site of SB202190.

  16. Expression of androgen-binding protein (ABP) in human cardiac myocytes.

    Science.gov (United States)

    Schock, H W; Herbert, Z; Sigusch, H; Figulla, H R; Jirikowski, G F; Lotze, U

    2006-04-01

    Cardiomyocytes are known to be androgen targets. Changing systemic steroid levels are thought to be linked to various cardiac ailments, including dilated cardiomyopathy (DCM). The mode of action of gonadal steroid hormones on the human heart is unknown to date. In the present study, we used high-resolution immunocytochemistry on semithin sections (1 microm thick), IN SITU hybridization, and mass spectrometry to investigate the expression of androgen-binding protein (ABP) in human myocardial biopsies taken from male patients with DCM. We observed distinct cytoplasmic ABP immunoreactivity in a fraction of the myocytes. IN SITU hybridization with synthetic oligonucleotide probes revealed specific hybridization signals in these cells. A portion of the ABP-positive cells contained immunostaining for androgen receptor. With SELDI TOF mass spectrometry of affinity purified tissue extracts of human myocardium, we confirmed the presence of a 50 kDa protein similar to ABP. Our observations provide evidence of an intrinsic expression of ABP in human heart. ABP may be secreted from myocytes in a paracrine manner perhaps to influence the bioavailabity of gonadal steroids in myocardium.

  17. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.

    Science.gov (United States)

    de Laat, W L; Appeldoorn, E; Sugasawa, K; Weterings, E; Jaspers, N G; Hoeijmakers, J H

    1998-08-15

    The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.

  18. Tumor necrosis factor-alpha inhibits differentiation of myogenic cells in human urethral rhabdosphincter.

    Science.gov (United States)

    Shinohara, Mayuka; Sumino, Yasuhiro; Sato, Fuminori; Kiyono, Tohru; Hashimoto, Naohiro; Mimata, Hiromitsu

    2017-06-01

    To examine the inhibitory effects of tumor necrosis factor-α on myogenic differentiation of human urethral rhabdosphincter cells. A rhabdosphincter sample was obtained from a patient who underwent total cystectomy. To expand the lifespan of the primary cultured cells, rhabdosphincter myogenic cells were immortalized with mutated cyclin-dependent kinase 4, cyclin D1 and telomerase. The differential potential of the cells was investigated. The transfected human rhabdosphincter cells were induced for myogenic differentiation with recombinant human tumor necrosis factor-α and/or the tumor necrosis factor-α antagonist etanercept at different concentrations, and activation of signaling pathways was monitored. Human rhabdosphincter cells were selectively cultured for at least 40 passages. Molecular analysis confirmed the expression of myosin heavy chain, which is a specific marker of differentiated muscle cells, significantly increased after differentiation induction. Although tumor necrosis factor-α treatment reduced the myosin heavy chain expression in a concentration-dependent manner, etanercept inhibited this suppression. Tumor necrosis factor-α suppressed phosphorylation of protein kinase B and p38, whereas etanercept pretreatment promoted phosphorylation and myosin heavy chain expression in a concentration-dependent manner. Tumor necrosis factor-α inhibits differentiation of urethral rhabdosphincter cells in part through the p38 mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Inhibition of tumor necrosis factor-α might be a useful strategy to treat stress urinary incontinence. © 2017 The Japanese Urological Association.

  19. Binding of α2-macroglobulin-thrombin complexes and methylamine-treated α2-macroglobulin to human blood monocytes

    International Nuclear Information System (INIS)

    Straight, D.L.; Jakoi, L.; McKee, P.A.; Snyderman, R.

    1988-01-01

    The binding of α 2 -macroglobulin (α 2 M) to human peripheral blood monocytes was investigated. Monocytes, the precursors of tissue macrophages, were isolated from fresh blood by centrifugal elutriation or density gradient centrifugation. Binding studies were performed using 125 I-labeled α 2 M. Cells and bound ligand were separated from free ligand by rapid vacuum filtration. Nonlinear least-squares analysis of data obtained in direct binding studies at 0 0 C showed that monocytes bound the α 2 M-thrombin complex with a K/sub d/ 3.0 +- .09 nM and the monocyte had 1545 +- 153 sitescell. Thrombin alone did not compete for the site. Binding was divalent cation dependent. Direct binding studies also demonstrated that monocytes bound methylamine-treated α 2 M in a manner similar to α 2 M-thrombin. Competitive binding studies showed that α 2 M-thrombin and methylamine-treated α 2 M bound to the same sites on the monocyte. In contrast, native α 2 M did not compete with α 2 M-thrombin for the site. Studies done at 37 0 C suggested that after binding, the monocyte internalized and degraded α 2 M-thrombin and excreted the degradation products. Receptor turnover and degradation of α 2 M-thrombin complexes were blocked in monocytes treated with chloroquine, an inhibitor of lysosomal function. The results indicate that human monocytes have a divalent cation dependent, high-affinity binding site for α 2 M-thrombin and methylamine-treated α 2 M which may function to clear α 2 M-proteinase complexes from the circulation

  20. The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing

    Science.gov (United States)

    Fang, Qing; Indzhykulian, Artur A; Mustapha, Mirna; Riordan, Gavin P; Dolan, David F; Friedman, Thomas B; Belyantseva, Inna A; Frolenkov, Gregory I; Camper, Sally A; Bird, Jonathan E

    2015-01-01

    The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture. DOI: http://dx.doi.org/10.7554/eLife.08627.001 PMID:26302205

  1. Human serum albumin unfolding pathway upon drug binding: A thermodynamic and spectroscopic description

    International Nuclear Information System (INIS)

    Cheema, Mohammad Arif; Taboada, Pablo; Barbosa, Silvia; Juarez, Josue; Gutierrez-Pichel, Manuel; Siddiq, Mohammad; Mosquera, Victor

    2009-01-01

    The interest on phenothiazine drugs has been increased during last years due to their proved utility in the treatment of several diseases and biomolecular processes. In the present work, the binding of the amphiphilic phenothiazines promazine and thioridazine hydrochlorides to the carrier protein human serum albumin (HSA) has been examined by ζ-potential, isothermal titration calorimetry (ITC), fluorescence and circular dichorism (CD) spectroscopies, and dynamic light scattering (DLS) at physiological pH with the aim of analyzing the role of the different interactions in the drug complexation process with this protein. The ζ-potential results were used to check the existence of complexation. This is confirmed by a progressive screening of the protein charge up to a reversal point as a consequence of drug binding. On the other hand, binding causes alterations on the tertiary and secondary structures of the protein, which were observed by fluorescence and CD spectroscopies, involving a two-step, three-state transition. The thermodynamics of the binding process was derived from ITC results. The binding enthalpies were negative, which reveal the existence of electrostatic interactions between protein and drug molecules. In addition, increases in entropy are consistent with the predominance of hydrophobic interactions. Two different classes of binding sites were detected, viz. Binding to the first class of binding sites is dominated by an enthalpic contribution due to electrostatic interactions whereas binding to a second class of binding sites is dominated by hydrophobic bonding. In the light of these results, protein conformational change resembles the acid-induced denaturation of HSA with accumulation of an intermediate state. Binding isotherms were derived from microcalorimetric results by using a theoretical model based on the Langmuir isotherm. On the other hand, the population distribution of the different species in solution and their sizes were determined

  2. Human serum albumin unfolding pathway upon drug binding: A thermodynamic and spectroscopic description

    Energy Technology Data Exchange (ETDEWEB)

    Cheema, Mohammad Arif [Grupo de Fisica de Coloides y Polimeros, Departamento de Fisica de la Materia Condensada, Facultad de Fisica, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela (Spain); Department of Chemistry, Quaid-i-Azam University, Islamabad 45320 (Pakistan); Taboada, Pablo [Grupo de Fisica de Coloides y Polimeros, Departamento de Fisica de la Materia Condensada, Facultad de Fisica, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela (Spain); Department of Chemistry, Quaid-i-Azam University, Islamabad 45320 (Pakistan)], E-mail: pablo.taboada@usc.es; Barbosa, Silvia; Juarez, Josue; Gutierrez-Pichel, Manuel [Grupo de Fisica de Coloides y Polimeros, Departamento de Fisica de la Materia Condensada, Facultad de Fisica, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela (Spain); Department of Chemistry, Quaid-i-Azam University, Islamabad 45320 (Pakistan); Siddiq, Mohammad [Department of Chemistry, Quaid-i-Azam University, Islamabad 45320 (Pakistan); Mosquera, Victor [Grupo de Fisica de Coloides y Polimeros, Departamento de Fisica de la Materia Condensada, Facultad de Fisica, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela (Spain); Department of Chemistry, Quaid-i-Azam University, Islamabad 45320 (Pakistan)

    2009-04-15

    The interest on phenothiazine drugs has been increased during last years due to their proved utility in the treatment of several diseases and biomolecular processes. In the present work, the binding of the amphiphilic phenothiazines promazine and thioridazine hydrochlorides to the carrier protein human serum albumin (HSA) has been examined by {zeta}-potential, isothermal titration calorimetry (ITC), fluorescence and circular dichorism (CD) spectroscopies, and dynamic light scattering (DLS) at physiological pH with the aim of analyzing the role of the different interactions in the drug complexation process with this protein. The {zeta}-potential results were used to check the existence of complexation. This is confirmed by a progressive screening of the protein charge up to a reversal point as a consequence of drug binding. On the other hand, binding causes alterations on the tertiary and secondary structures of the protein, which were observed by fluorescence and CD spectroscopies, involving a two-step, three-state transition. The thermodynamics of the binding process was derived from ITC results. The binding enthalpies were negative, which reveal the existence of electrostatic interactions between protein and drug molecules. In addition, increases in entropy are consistent with the predominance of hydrophobic interactions. Two different classes of binding sites were detected, viz. Binding to the first class of binding sites is dominated by an enthalpic contribution due to electrostatic interactions whereas binding to a second class of binding sites is dominated by hydrophobic bonding. In the light of these results, protein conformational change resembles the acid-induced denaturation of HSA with accumulation of an intermediate state. Binding isotherms were derived from microcalorimetric results by using a theoretical model based on the Langmuir isotherm. On the other hand, the population distribution of the different species in solution and their sizes were

  3. Zinc binding activity of human metapneumovirus M2-1 protein is indispensable for viral replication and pathogenesis in vivo.

    Science.gov (United States)

    Cai, Hui; Zhang, Yu; Ma, Yuanmei; Sun, Jing; Liang, Xueya; Li, Jianrong

    2015-06-01

    Human metapneumovirus (hMPV) is a member of the Pneumovirinae subfamily in the Paramyxoviridae family that causes respiratory tract infections in humans. Unlike members of the Paramyxovirinae subfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional antitermination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesis in vivo. These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes. The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute

  4. Potent radiolabeled human renin inhibitor, [3H]SR42128: enzymatic, kinetic, and binding studies to renin and other aspartic proteases

    International Nuclear Information System (INIS)

    Cumin, F.; Nisato, D.; Gagnol, J.P.; Corvol, P.

    1987-01-01

    The in vitro binding of [ 3 H]SR42128 (Iva-Phe-Nle-Sta-Ala-Sta-Arg), a potent inhibitor of human renin activity, to purified human renin and a number of other aspartic proteases was examined. SR42128 was found to be a competitive inhibitor of human renin, with a K/sub i/ of 0.35 nM at pH 5.7 and 2.0 nM at pH 7.4; it was thus more effective at pH 5.7 than at pH 7.4. Scatchard analysis of the interaction binding of [ 3 H]SR42128 to human renin indicated that binding was reversible and saturable at both pH 5.7 and pH 7.4. There was a single class of binding sites, and the K/sub D/ was 0.9 nM at pH 5.7 and 1 nM at pH 7.4. The association rate was 10 times more rapid at pH 5.7 than at pH 7.4, but there was no difference between the rates of dissociation of the enzyme-inhibitor complex at the two pHs. The effect of pH on the binding of [ 3 H]SR42128 to human renin, cathepsin D, pepsin, and gastricsin was also examined over the pH range 3-8. All the aspartic proteases had a high affinity for the inhibitor at low pH. However, at pH 7.4, [ 3 H]SR42128 was bound only to human renin and to none of the other aspartic proteases. Competitive binding studies with [ 3 H]SR42128 and a number of other inhibitors on human renin or cathepsin D were used to examine the relationships between structure and activity in these systems. The study as a whole indicates that pH plays a major role in the binding of [ 3 H]SR42128 to aspartic proteases and that the nature of the inhibitor residue reacting with the renin S 2 subsites is of critical importance for the specificity of the renin-inhibitor interaction

  5. Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lianying [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); College of Life Science, Dezhou University, Dezhou 253023 (China); Ren, Xiao-Min; Wan, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China)

    2014-09-15

    Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.

  6. Differential proteome analysis of human embryonic kidney cell line (HEK-293 following mycophenolic acid treatment

    Directory of Open Access Journals (Sweden)

    Rahman Hazir

    2011-09-01

    Full Text Available Abstract Background Mycophenolic acid (MPA is widely used as a post transplantation medicine to prevent acute organ rejection. In the present study we used proteomics approach to identify proteome alterations in human embryonic kidney cells (HEK-293 after treatment with therapeutic dose of MPA. Following 72 hours MPA treatment, total protein lysates were prepared, resolved by two dimensional gel electrophoresis and differentially expressed proteins were identified by QTOF-MS/MS analysis. Expressional regulations of selected proteins were further validated by real time PCR and Western blotting. Results The proliferation assay demonstrated that therapeutic MPA concentration causes a dose dependent inhibition of HEK-293 cell proliferation. A significant apoptosis was observed after MPA treatment, as revealed by caspase 3 activity. Proteome analysis showed a total of 12 protein spots exhibiting differential expression after incubation with MPA, of which 7 proteins (complement component 1 Q subcomponent-binding protein, electron transfer flavoprotein subunit beta, cytochrome b-c1 complex subunit, peroxiredoxin 1, thioredoxin domain-containing protein 12, myosin regulatory light chain 2, and profilin 1 showed significant increase in their expression. The expression of 5 proteins (protein SET, stathmin, 40S ribosomal protein S12, histone H2B type 1 A, and histone H2B type 1-C/E/F/G/I were down-regulated. MPA mainly altered the proteins associated with the cytoskeleton (26%, chromatin structure/dynamics (17% and energy production/conversion (17%. Both real time PCR and Western blotting confirmed the regulation of myosin regulatory light chain 2 and peroxiredoxin 1 by MPA treatment. Furthermore, HT-29 cells treated with MPA and total kidney cell lysate from MMF treated rats showed similar increased expression of myosin regulatory light chain 2. Conclusion The emerging use of MPA in diverse pathophysiological conditions demands in-depth studies to

  7. The IQ motif drives the nuclear translocation of nuclear myosin I

    Czech Academy of Sciences Publication Activity Database

    Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Hozák, Pavel

    2008-01-01

    Roč. 275, č. 1 (2008), s. 67-67 E-ISSN 1742-4658. [FEBS Congress /33rd/, IUBMB conference /11th/. 28.06.2008-03.07.2008, Athens] R&D Projects: GA MŠk LC545; GA ČR(CZ) GA204/07/1592 Grant - others:GAČR(CZ) GD204/05/H023 Program:GD Institutional research plan: CEZ:AV0Z50520514 Keywords : nuclear myosin * nuclear transport Subject RIV: EB - Genetics ; Molecular Biology

  8. Characterizing Active Pharmaceutical Ingredient Binding to Human Serum Albumin by Spin-Labeling and EPR Spectroscopy.

    Science.gov (United States)

    Hauenschild, Till; Reichenwallner, Jörg; Enkelmann, Volker; Hinderberger, Dariush

    2016-08-26

    Drug binding to human serum albumin (HSA) has been characterized by a spin-labeling and continuous-wave (CW) EPR spectroscopic approach. Specifically, the contribution of functional groups (FGs) in a compound on its albumin-binding capabilities is quantitatively described. Molecules from different drug classes are labeled with EPR-active nitroxide radicals (spin-labeled pharmaceuticals (SLPs)) and in a screening approach CW-EPR spectroscopy is used to investigate HSA binding under physiological conditions and at varying ratios of SLP to protein. Spectral simulations of the CW-EPR spectra allow extraction of association constants (KA ) and the maximum number (n) of binding sites per protein. By comparison of data from 23 SLPs, the mechanisms of drug-protein association and the impact of chemical modifications at individual positions on drug uptake can be rationalized. Furthermore, new drug modifications with predictable protein binding tendency may be envisaged. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts.

    Science.gov (United States)

    Kasahara, Kohji; Kaneda, Mizuho; Miki, Toshiaki; Iida, Kazuko; Sekino-Suzuki, Naoko; Kawashima, Ikuo; Suzuki, Hidenori; Shimonaka, Motoyuki; Arai, Morio; Ohno-Iwashita, Yoshiko; Kojima, Soichi; Abe, Mitsuhiro; Kobayashi, Toshihide; Okazaki, Toshiro; Souri, Masayoshi; Ichinose, Akitada; Yamamoto, Naomasa

    2013-11-07

    Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion pr