Sample records for ideal cleavage strength

  1. Ab initio calculations of ideal strength and lattice instability in W-Ta and W-Re alloys

    NASA Astrophysics Data System (ADS)

    Yang, Chaoming; Qi, Liang

    2018-01-01

    An important theoretical criterion to evaluate the ductility of metals with a body-centered cubic (bcc) lattice is the mechanical failure mode of their perfect crystals under tension along <;100 >; directions. When the tensile stress reaches the ideal tensile strength, the pure W crystal fails by a cleavage fracture along the {100 } plane so that it is intrinsically brittle. To discover the strategy to improve its ductility, we performed density functional theory and density functional perturbation theory calculations to study the ideal tensile strength and the lattice instability under <100 > tension for both W-Ta and W-Re alloys. Anisotropic linear elastic fracture mechanics (LEFM) theory and Rice's criterion were also applied to analyze the mechanical instability at the crack tip under <100 > tension based on the competition between cleavage propagation and dislocation emission. The results show that the intrinsic ductility can be achieved in both W-Ta and W-Re, however, by different mechanisms. Even though W-Ta alloys with low Ta concentrations are still intrinsically brittle, the intrinsic ductility of W-Ta alloys with high Ta concentrations is promoted by elastic shear instability before the cleavage failure. The intrinsic ductility of W-Re alloys is produced by unstable transverse phonon waves before the cleavage failure, and the corresponding phonon mode is related to the generation of 1/2 <111 > {2 ¯11 } dislocation in bcc crystals. The ideal tensile calculations, phonon analyses, and anisotropic LEFM examinations are mutually consistent in the evaluation of intrinsic ductility. These results bring us physical insights on the ductility-brittle mechanisms of W alloys under extreme stress conditions.

  2. Ideal strength of bcc molybdenum and niobium

    NASA Astrophysics Data System (ADS)

    Luo, Weidong; Roundy, D.; Cohen, Marvin L.; Morris, J. W.

    2002-09-01

    The behavior of bcc Mo and Nb under large strain was investigated using the ab initio pseudopotential density-functional method. We calculated the ideal shear strength for the {211}<111> and {011}<111> slip systems and the ideal tensile strength in the <100> direction, which are believed to provide the minimum shear and tensile strengths. As either material is sheared in either of the two systems, it evolves toward a stress-free tetragonal structure that defines a saddle point in the strain-energy surface. The inflection point on the path to this tetragonal ``saddle-point'' structure sets the ideal shear strength. When either material is strained in tension along <100>, it initially follows the tetragonal, ``Bain,'' path toward a stress-free fcc structure. However, before the strained crystal reaches fcc, its symmetry changes from tetragonal to orthorhombic; on continued strain it evolves toward the same tetragonal saddle point that is reached in shear. In Mo, the symmetry break occurs after the point of maximum tensile stress has been passed, so the ideal strength is associated with the fcc extremum as in W. However, a Nb crystal strained in <100> becomes orthorhombic at tensile stress below the ideal strength. The ideal tensile strength of Nb is associated with the tetragonal saddle point and is caused by failure in shear rather than tension. In dimensionless form, the ideal shear and tensile strengths of Mo (τ*=τm/G111=0.12, σ*=σm/E100=0.078) are essentially identical to those previously calculated for W. Nb is anomalous. Its dimensionless shear strength is unusually high, τ*=0.15, even though the saddle-point structure that causes it is similar to that in Mo and W, while its dimensionless tensile strength, σ*=0.079, is almost the same as that of Mo and W, even though the saddle-point structure is quite different.

  3. Cleavage fracture in high strength low alloy weld metal

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bose, W.W.; Bowen, P.; Strangwood, M.

    1996-12-31

    The present investigation gives an evaluation of the effect of microstructure on the cleavage fracture process of High Strength Low Alloy (HSLA) multipass weld metals. With additions of alloying elements, such as Ti, Ni, Mo and Cr, the microstructure of C-Mn weld metal changes from the classical composition, i.e., allotriomorphic ferrite with acicular ferrite and Widmanstaetten ferrite, to bainite and low carbon martensite. Although the physical metallurgy of some HSLA weld metals has been studied before, more work is necessary to correlate the effect of the microstructure on the fracture behavior of such weld metals. In this work detailed microstructuralmore » analysis was carried out using optical and electron (SEM and TEM) microscopy. Single edge notched (SEN) bend testpieces were used to assess the cleavage fracture stress, {sigma}{sub F}. Inclusions beneath the notch surface were identified as the crack initiators of unstable cleavage fracture. From the size of such inclusions and the value of tensile stress predicted at the initiation site, the effective surface energy for cleavage was calculated using a modified Griffth energy balance for a penny shape crack. The results suggest that even though inclusions initiate cleavage fracture, the local microstructure may play an important role in the fracture process of these weld metals. The implications of these observations for a quantitative theory of the cleavage fracture of ferritic steels is discussed.« less

  4. Handgrip Strength and Ideal Cardiovascular Health among Colombian Children and Adolescents.

    PubMed

    Ramírez-Vélez, Robinson; Tordecilla-Sanders, Alejandra; Correa-Bautista, Jorge Enrique; Peterson, Mark D; Garcia-Hermoso, Antonio

    2016-12-01

    To evaluate the association between handgrip strength and ideal cardiovascular health (CVH) in Colombian children and adolescents. During the 2014-2015 school years, we examined a cross-sectional component of the FUPRECOL (Association for Muscular Strength with Early Manifestation of Cardiovascular Disease Risk Factors among Colombian Children and Adolescents) study. Participants included 1199 (n = 627 boys) youths from Bogota (Colombia). Handgrip strength was measured with a standard adjustable hand held dynamometer and expressed relative to body mass (handgrip/body mass) and as absolute values in kilograms. Ideal CVH, as defined by the American Heart Association, was determined as meeting ideal levels of the following components: 4 behaviors (smoking status, body mass index, cardiorespiratory fitness, and diet) and 3 factors (total cholesterol, blood pressure, and glucose). Higher levels of handgrip strength (both absolute and relative values) were associated with a higher frequency of ideal CVH metrics in both sexes (P for trend ≤ .001). Also, higher levels of handgrip strength were associated with a greater number of ideal health behaviors (P for trend < .001 in both boys and girls), and with a higher number of ideal health factors in boys (P for trend < .001). Finally, levels of handgrip strength were similar between ideal versus nonideal glucose or total cholesterol groups in girls. Handgrip strength was strongly associated with ideal CVH in Colombian children and adolescents, and thus supports the relevance of early targeted interventions to promote strength adaptation and preservation as part of primordial prevention. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Extended Hall-Petch Relationships for Yield, Cleavage and Intergranular Fracture Strengths of bcc Steel and Its Deformation and Fracture Behaviors

    NASA Astrophysics Data System (ADS)

    Heo, N. H.; Heo, Y.-U.; Kwon, S. K.; Kim, N. J.; Kim, S.-J.; Lee, H.-C.

    2018-03-01

    Extended Hall-Petch relationships for yield ( σy ), cleavage ( σ_{cl} ) and intergranular fracture ( σ_{ig} ) strengths of pure iron have been established through the direct calculation of the proportional constant (k) and the estimation of the friction stress (σ0 ) . The magnitude orders of k and σ0 are generally ky < k_{cl} < k_{ig} and σ_{y0} < σ_{cl0} < σ_{ig0} , respectively. Based on the Hall-Petch relationships, micro-yielding in a bcc steel occurs at the instance that the pile-up dislocations within a specific grain showing the Schmid factor of 0.5 propagate into the neighboring grain. The initial brittle crack is formed at the instance that the flow strength exceeds the brittle fracture strength. Once the brittle crack is formed, it grows catastrophically. Due to the smallest and ky and σ_{y0} , the cleavage and the intergranular fracture occur always after micro-yielding. The {100} cleavage fracture of the steel is due to the lowest theoretical {100} cleavage strength. Due to the thermal components included in cleavage and intergranular fracture strengths, they show also the temperature and strain rate dependence observed in yield strength. The increase in susceptibility to brittle fracture with decreasing temperature and increasing strain rate is due to the increase in dislocation density which causes the high work hardening rate.

  6. Fracture Strength of AlLiB14

    NASA Astrophysics Data System (ADS)

    Wan, L. F.; Beckman, S. P.

    2012-10-01

    The orthorhombic boride crystal family XYB14, where X and Y are metal atoms, plays a critical role in a unique class of superhard compounds, yet there have been no studies aimed at understanding the origin of the mechanical strength of this compound. We present here the results from a comprehensive investigation into the fracture strength of the archetypal AlLiB14 crystal. First principles, ab initio, methods are used to determine the ideal brittle cleavage strength for several high-symmetry orientations. The elastic tensor and the orientation-dependent Young’s modulus are calculated. From these results the lower bound fracture strength of AlLiB14 is predicted to be between 29 and 31 GPa, which is near the measured hardness reported in the literature. These results indicate that the intrinsic strength of AlLiB14 is limited by the interatomic B-B bonds that span between the B layers.

  7. Improving the toughness of ultrahigh strength steel

    NASA Astrophysics Data System (ADS)

    Sato, Koji

    2002-01-01

    The ideal structural steel combines high strength with high fracture toughness. This dissertation discusses the toughening mechanism of the Fe/Co/Ni/Cr/Mo/C steel, AerMet 100, which has the highest toughness/strength combination among all commercial ultrahigh strength steels. The possibility of improving the toughness of this steel was examined by considering several relevant factors. Chapter 1 reviews the mechanical properties of ultrahigh strength steels and the physical metallurgy of AerMet 100. It also describes the fracture mechanisms of steel, i.e. ductile microvoid coalescence, brittle transgranular cleavage, and intergranular separation. Chapter 2 examines the strength-toughness relationship for three heats of AerMet 100. A wide variation of toughness is obtained at the same strength level. The toughness varies despite the fact that all heat fracture in the ductile fracture mode. The difference originates from the inclusion content. Lower inclusion volume fraction and larger inclusion spacing gives rise to a greater void growth factor and subsequently a higher fracture toughness. The fracture toughness value, JIc, is proportional to the particle spacing of the large non-metallic inclusions. Chapter 3 examines the ductile-brittle transition of AerMet 100 and the effect of a higher austenitization temperature, using the Charpy V-notch test. The standard heat treatment condition of AerMet 100 shows a gradual ductile-brittle transition due to its fine effective grain size. Austenitization at higher temperature increases the prior austenite grain size and packet size, leading to a steeper transition at a higher temperature. Both transgranular cleavage and intergranular separation are observed in the brittle fracture mode. Chapter 4 examines the effect of inclusion content, prior austenite grain size, and the amount of austenite on the strength-toughness relationship. The highest toughness is achieved by low inclusion content, small prior austenite grain size

  8. Transition-metal alloying of γ'-Ni3Al : Effects on the ideal uniaxial compressive strength from first-principles calculations

    NASA Astrophysics Data System (ADS)

    Wen, Minru; Wang, Chong-Yu

    2018-01-01

    The addition of transition-metal (TM) elements into the γ' precipitate phase of a Ni-based single-crystal superalloy can significantly affect its mechanical properties, including the intrinsic mechanical property of compressive strength. Using first-principles density functional calculations, the effects of 3 d (Sc-Zn), 4 d (Y-Cd), and 5 d (Hf-Au) TM alloying elements on the ideal uniaxial compressive strength of γ'-Ni3Al were investigated. The stress-strain relationships of pure Ni3Al under [100], [110], and [111] compressive loads and the site occupancy behavior of TM elements in Ni3Al were previously studied using a total-energy method based on density functional theory. Our results showed that the capacity of TM elements for strengthening the ideal compressive strength was associated with the d -electron number. The alloying elements with half-filled d bands (i.e., Cr, Mo, W, Tc, and Re) manifested the greatest efficacy for improving the ideal strength of Ni3Al under a deformation along the weakest compressive direction. Furthermore, the charge redistribution of Ni3Al doped with 5 d elements were also analyzed to understand the strengthening mechanisms of TM elements in the γ'-Ni3Al phase.

  9. On the temperature independence of statistical model parameters for cleavage fracture in ferritic steels

    NASA Astrophysics Data System (ADS)

    Qian, Guian; Lei, Wei-Sheng; Niffenegger, M.; González-Albuixech, V. F.

    2018-04-01

    The work relates to the effect of temperature on the model parameters in local approaches (LAs) to cleavage fracture. According to a recently developed LA model, the physical consensus of plastic deformation being a prerequisite to cleavage fracture enforces any LA model of cleavage fracture to observe initial yielding of a volume element as its threshold stress state to incur cleavage fracture in addition to the conventional practice of confining the fracture process zone within the plastic deformation zone. The physical consistency of the new LA model to the basic LA methodology and the differences between the new LA model and other existing models are interpreted. Then this new LA model is adopted to investigate the temperature dependence of LA model parameters using circumferentially notched round tensile specimens. With the published strength data as input, finite element (FE) calculation is conducted for elastic-perfectly plastic deformation and the realistic elastic-plastic hardening, respectively, to provide stress distributions for model calibration. The calibration results in temperature independent model parameters. This leads to the establishment of a 'master curve' characteristic to synchronise the correlation between the nominal strength and the corresponding cleavage fracture probability at different temperatures. This 'master curve' behaviour is verified by strength data from three different steels, providing a new path to calculate cleavage fracture probability with significantly reduced FE efforts.

  10. Temperature-dependent ideal strength and stacking fault energy of fcc Ni: a first-principles study of shear deformation.

    PubMed

    Shang, S L; Wang, W Y; Wang, Y; Du, Y; Zhang, J X; Patel, A D; Liu, Z K

    2012-04-18

    Variations of energy, stress, and magnetic moment of fcc Ni as a response to shear deformation and the associated ideal shear strength (τ(IS)), intrinsic (γ(SF)) and unstable (γ(US)) stacking fault energies have been studied in terms of first-principles calculations under both the alias and affine shear regimes within the {111} slip plane along the <112> and <110> directions. It is found that (i) the intrinsic stacking fault energy γ(SF) is nearly independent of the shear deformation regimes used, albeit a slightly smaller value is predicted by pure shear (with relaxation) compared to the one from simple shear (without relaxation); (ii) the minimum ideal shear strength τ(IS) is obtained by pure alias shear of {111}<112>; and (iii) the dissociation of the 1/2[110] dislocation into two partial Shockley dislocations (1/6[211] + 1/6[121]) is observed under pure alias shear of {111}<110>. Based on the quasiharmonic approach from first-principles phonon calculations, the predicted γ(SF) has been extended to finite temperatures. In particular, using a proposed quasistatic approach on the basis of the predicted volume versus temperature relation, the temperature dependence of τ(IS) is also obtained. Both the γ(SF) and the τ(IS) of fcc Ni decrease with increasing temperature. The computed ideal shear strengths as well as the intrinsic and unstable stacking fault energies are in favorable accord with experiments and other predictions in the literature.

  11. Cleavage fracture in pearlitic eutectoid steel

    NASA Astrophysics Data System (ADS)

    Alexander, D. J.; Bernstein, I. M.

    1989-11-01

    The effect of microstructure on flow and fracture properties of fully pearlitic steel has been studied by independently varying the prior austenite grain size and the pearlite interlamellar spacing through appropriate heat treatments. The yield strength is independent of the prior austenite grain size but increases as the interlamellar spacing or the temperature decreases. The microstructural dependence can be explained by using a model which assumes that yielding is controlled by dislocation motion in the ferrite lamellae. The critical tensile stress for cleavage fracture is found to be independent of prior austenite grain size, increasing as the interlamellar spacing decreases. The cleavage fracture stress is independent of temperature for fine pearlite but increases as the temperature decreases for coarse pearlite. The associated fracture in blunt notch specimens initiates at inclusions beneath notch surface near the location of maximum tensile stress. From the size of such inclusions, the effective surface energy for cleavage fracture can be directly calculated and is found to be independent of temperature and prior austenite grain size but to increase as the interlamellar spacing decreases, from about 5 to 13 J/m2 for the range of microstructures and temperatures used in this study. Additional measurements of the effective surface energy and further theoretical analyses of the cleavage process are needed.

  12. The fractography-modeling link in cleavage fracture

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thompson, A.W.

    1997-12-31

    Cleavage fracture has historically been modelled, out of necessity, in rather idealized terms. In real materials, however, there are a number of difficulties in linking such models with metallographic and fractographic observations. Some of the most vivid examples occur for {alpha}{sub 2} titanium aluminide alloys, in which, when the microstructure contains primary {alpha}{sub 2} particles, the primary particles crack first. When basketweave or Widmanstaetten structures of {alpha}{sub 2} laths comprise the microstructure, it appears that individual laths crack first. And in colony structures, cracking occurs first across the {alpha}{sub 2} lath colonies. Both detailed fractographic observations, and also a statisticalmore » model for brittle fracture by failure of weakest links, have been developed. The extent to which this can be interpreted in classical cleavage terms will be discussed.« less

  13. Reducing the Ideal Shear Strengths of ZrB2 by High Efficient Alloying Elements (Ag, Au, Pd and Pt)

    NASA Astrophysics Data System (ADS)

    Dai, Fu-Zhi; Zhou, Yanchun

    2017-02-01

    Activating the plasticity of ZrB2 is a promising approach to improve its key properties for applications in hypersonic vehicles, including high temperature strength and thermal shock resistance. The present work demonstrates that ideal shear strength of ZrB2, which is a good indicator of the critical stress for dislocation nucleation, can be significantly reduced by dissolving of appropriate alloying elements. Analyzing on the bonding nature of ZrB2 reveals that choosing alloying elements with low energy valence electrons will prevent electron transferring from alloying element to the electron deficient B-B π orbits, which will reduce the local stability of the region surrounding the alloying element. Under the criterion, elements with d electrons tending to be full-filled (Ag, Au, Pd and Pt, the full-filled state is associated with low energy level) are selected as promising candidates with their prominent efficiency in reducing ideal shear strengths verified by first-principles calculations. The results provide useful guidelines for further designs of ZrB2 based materials, especially for improving their mechanical properties.

  14. Reducing the Ideal Shear Strengths of ZrB2 by High Efficient Alloying Elements (Ag, Au, Pd and Pt)

    PubMed Central

    Dai, Fu-Zhi; Zhou, Yanchun

    2017-01-01

    Activating the plasticity of ZrB2 is a promising approach to improve its key properties for applications in hypersonic vehicles, including high temperature strength and thermal shock resistance. The present work demonstrates that ideal shear strength of ZrB2, which is a good indicator of the critical stress for dislocation nucleation, can be significantly reduced by dissolving of appropriate alloying elements. Analyzing on the bonding nature of ZrB2 reveals that choosing alloying elements with low energy valence electrons will prevent electron transferring from alloying element to the electron deficient B-B π orbits, which will reduce the local stability of the region surrounding the alloying element. Under the criterion, elements with d electrons tending to be full-filled (Ag, Au, Pd and Pt, the full-filled state is associated with low energy level) are selected as promising candidates with their prominent efficiency in reducing ideal shear strengths verified by first-principles calculations. The results provide useful guidelines for further designs of ZrB2 based materials, especially for improving their mechanical properties. PMID:28233838

  15. Reducing the Ideal Shear Strengths of ZrB2 by High Efficient Alloying Elements (Ag, Au, Pd and Pt).

    PubMed

    Dai, Fu-Zhi; Zhou, Yanchun

    2017-02-24

    Activating the plasticity of ZrB 2 is a promising approach to improve its key properties for applications in hypersonic vehicles, including high temperature strength and thermal shock resistance. The present work demonstrates that ideal shear strength of ZrB 2 , which is a good indicator of the critical stress for dislocation nucleation, can be significantly reduced by dissolving of appropriate alloying elements. Analyzing on the bonding nature of ZrB 2 reveals that choosing alloying elements with low energy valence electrons will prevent electron transferring from alloying element to the electron deficient B-B π orbits, which will reduce the local stability of the region surrounding the alloying element. Under the criterion, elements with d electrons tending to be full-filled (Ag, Au, Pd and Pt, the full-filled state is associated with low energy level) are selected as promising candidates with their prominent efficiency in reducing ideal shear strengths verified by first-principles calculations. The results provide useful guidelines for further designs of ZrB 2 based materials, especially for improving their mechanical properties.

  16. The Joint Strength and Fracture Mechanisms of TC4/TC4 and TA0/TA0 Brazed with Ti-25Cu-15Ni Braze Alloy

    NASA Astrophysics Data System (ADS)

    Zou, Zhihuan; Zeng, Fanhao; Wu, Haobo; Liu, Jian; Li, Yi; Gu, Yi; Yuan, Tiechui; Zhang, Fuqin

    2017-05-01

    In this paper, Ti-25Cu-15Ni (mass ratio) braze alloys were prepared by vacuum arc melting. Additionally, the TA0 pure titanium and TC4 titanium alloy were brazed with the Ti-25Cu-15Ni braze alloy at 960, 980, 1000, 1020, and 1040 °C. The effects of the braze temperature on the tensile strength of the TA0 and TC4 joints and their fracture mechanisms were studied. The maximum tensile strength of the TA0 joints of 219.9 ± 0.1 MPa was achieved at a brazing temperature of 980 °C, and the maximum tensile strength of the TC4 joints of 832.9 ± 0.1 MPa was achieved at the same brazing temperature. These results indicate that their ideal joint strength is comparable. According to the fractography results of the TA0 joints, a mixed fracture morphology is indicated. The TA0 fracture surface is dominated by cleavage fracture with a small contribution from ductile fracture. The TC4 joint fracture arises from cleavage.

  17. Experimental verification of cleavage characteristic stress vs grain size

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lei, W.; Li, D.; Yao, M.

    Instead of the accepted cleavage fracture stress [sigma][sub f] proposed by Knott et al, a new parameter S[sub co], named as ''cleavage characteristic stress,'' has been recently recommended to characterize the microscopic resistance to cleavage fracture. To give a definition, S[sub co] is the fracture stress at the brittle/ductile transition temperature of steels in plain tension, below which the yield strength approximately equals the true fracture stress combined with an abrupt curtailment of ductility. By considering a single-grain microcrack arrested at a boundary, Huang and Yao set up an expression of S[sub co] as a function of grain size. Themore » present work was arranged to provide an experimental verification of S[sub co] vs grain size.« less

  18. Intra- and Interdimeric Caspase-8 Self-Cleavage Controls Strength and Timing of CD95-Induced Apoptosis

    PubMed Central

    Kallenberger, Stefan M.; Beaudouin, Joël; Claus, Juliane; Fischer, Carmen; Sorger, Peter K.; Legewie, Stefan; Eils, Roland

    2014-01-01

    Apoptosis in response to the ligand CD95L (also known as Fas ligand) is initiated by caspase-8, which is activated by dimerization and self-cleavage at death-inducing signaling complexes (DISCs). Previous work indicated that the degree of substrate cleavage by caspase-8 determines whether a cell dies or survives in response to a death stimulus. To determine how a death ligand stimulus is effectively translated into caspase-8 activity, we assessed this activity over time in single cells with compartmentalized probes that are cleaved by caspase-8, and used multiscale modeling to simultaneously describe single-cell and population data with an ensemble of single-cell models. We derived and experimentally validated a minimal model in which cleavage of caspase-8 in the enzymatic domain occurs in an interdimeric manner through interaction between DISCs, whereas prodomain cleavage sites are cleaved in an intradimeric manner within DISCs. Modeling indicated that sustained membrane-bound caspase-8 activity is followed by transient cytosolic activity, which can be interpreted as a molecular timer mechanism reflected by a limited lifetime of active caspase-8. The activation of caspase-8 by combined intra- and interdimeric cleavage ensures weak signaling at low concentrations of CD95L and strongly accelerated activation at higher ligand concentrations, thereby contributing to precise control of apoptosis. PMID:24619646

  19. Measurement of the cleavage energy of graphite

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Wen; Dai, Shuyang; Li, Xide

    Here, the basal plane cleavage energy (CE) of graphite is a key material parameter for understanding many of the unusual properties of graphite, graphene and carbon nanotubes. Nonetheless, a wide range of values for the CE has been reported and no consensus has yet emerged. Here we report the first direct, accurate experimental measurement of the CE of graphite using a novel method based on the self-retraction phenomenon in graphite. The measured value, 0.37±0.01 J m –2 for the incommensurate state of bicrystal graphite, is nearly invariant with respect to temperature (22 °C≤T≤198 °C) and bicrystal twist angle, and insensitivemore » to impurities from the atmosphere. The CE for the ideal ABAB graphite stacking, 0.39±0.02 J m –2, is calculated based on a combination of the measured CE and a theoretical calculation. These experimental measurements are also ideal for use in evaluating the efficacy of competing theoretical approaches.« less

  20. Measurement of the cleavage energy of graphite

    DOE PAGES

    Wang, Wen; Dai, Shuyang; Li, Xide; ...

    2015-08-28

    Here, the basal plane cleavage energy (CE) of graphite is a key material parameter for understanding many of the unusual properties of graphite, graphene and carbon nanotubes. Nonetheless, a wide range of values for the CE has been reported and no consensus has yet emerged. Here we report the first direct, accurate experimental measurement of the CE of graphite using a novel method based on the self-retraction phenomenon in graphite. The measured value, 0.37±0.01 J m –2 for the incommensurate state of bicrystal graphite, is nearly invariant with respect to temperature (22 °C≤T≤198 °C) and bicrystal twist angle, and insensitivemore » to impurities from the atmosphere. The CE for the ideal ABAB graphite stacking, 0.39±0.02 J m –2, is calculated based on a combination of the measured CE and a theoretical calculation. These experimental measurements are also ideal for use in evaluating the efficacy of competing theoretical approaches.« less

  1. The elastic stability, bifurcation and ideal strength of gold under hydrostatic stress: an ab initio calculation.

    PubMed

    Wang, Hao; Li, Mo

    2009-11-11

    In this paper, we employ an ab initio density functional theory calculation to investigate the elastic stability of face-centered cubic Au under hydrostatic deformation. We identify the elastic stiffness constant B(ijkl) as the coefficient in the stress-strain relation for an arbitrary deformed state, and use it to test the stability condition. We show that this criterion bears the same physics as that proposed earlier by Frenkel and Orowan and agrees with the Born-Hill criterion. The results from those two approaches agree well with each other. We show that the stability limit, or instability, of the perfect Au crystal under hydrostatic expansion is not associated with the bulk stiffness modulus as predicted in the previous work; rather it is caused by a shear instability associated with the vanishing rhombohedral shear stiffness modulus. The deviation of the deformation mode from the primary hydrostatic loading path signals a bifurcation or symmetry breaking in the ideal crystal. The corresponding ideal hydrostatic strength for Au is 19.2 GPa at the Lagrangian expansion strain of ∼0.06. In the case of compression, Au remains stable over the entire pressure range in our calculation.

  2. A set of simple cell processes is sufficient to model spiral cleavage.

    PubMed

    Brun-Usan, Miguel; Marín-Riera, Miquel; Grande, Cristina; Truchado-Garcia, Marta; Salazar-Ciudad, Isaac

    2017-01-01

    During cleavage, different cellular processes cause the zygote to become partitioned into a set of cells with a specific spatial arrangement. These processes include the orientation of cell division according to: an animal-vegetal gradient; the main axis (Hertwig's rule) of the cell; and the contact areas between cells or the perpendicularity between consecutive cell divisions (Sachs' rule). Cell adhesion and cortical rotation have also been proposed to be involved in spiral cleavage. We use a computational model of cell and tissue biomechanics to account for the different existing hypotheses about how the specific spatial arrangement of cells in spiral cleavage arises during development. Cell polarization by an animal-vegetal gradient, a bias to perpendicularity between consecutive cell divisions (Sachs' rule), cortical rotation and cell adhesion, when combined, reproduce the spiral cleavage, whereas other combinations of processes cannot. Specifically, cortical rotation is necessary at the 8-cell stage to direct all micromeres in the same direction. By varying the relative strength of these processes, we reproduce the spatial arrangement of cells in the blastulae of seven different invertebrate species. © 2017. Published by The Company of Biologists Ltd.

  3. Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

    PubMed Central

    Sheng, Gang; Zhao, Hongtu; Wang, Jiuyu; Rao, Yu; Tian, Wenwen; Swarts, Daan C.; van der Oost, John; Patel, Dinshaw J.; Wang, Yanli

    2014-01-01

    We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg2+ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand. PMID:24374628

  4. Can sucrose cleavage enzymes serve as markers for sink strength and is sucrose a signal molecule during plant sink development?

    Treesearch

    C.C. Black; T. Lobodia; J.-Q Chen; Shi-Jean S. Sung

    1995-01-01

    Sucrose cleavage is an essential reaction for higher plant cells to initiate intermediary metabolism and to direct its carbon into the host of essential compounds derived therefrom for maintaining the cells of intact plants.This review will focus on: the concentrations of sucrose available to plant cells; some biochemical traits of sucrose cleavage enzymes; the...

  5. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor L.; Brow, Mary Ann D.; Dahlberg, James E.

    2007-12-11

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  6. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow; Mary Ann D.; Dahlberg, James E.

    2010-11-09

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  7. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    2000-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  8. Characteristics of eyes with inner retinal cleavage.

    PubMed

    Hwang, Young Hoon; Kim, Yong Yeon; Kim, Hwang Ki; Sohn, Yong Ho

    2015-02-01

    Inner retinal cleavage can be misdiagnosed as a glaucomatous retinal nerve fiber layer (RNFL) defect. This study was performed to characterize eyes with inner retinal cleavage. Inner retinal cleavage is defined as the appearance of a dark spindle-shaped space between the nerve fibers. Patients who presented at our institution with inner retinal cleavage were enrolled in the study. All participants were evaluated by fundus examination, visual field testing with standard automated perimetry, and optical coherence tomography (OCT) imaging. A total of 15 eyes of 11 subjects with inner retinal cleavage were included in the study. The median age of the subjects was 57 years (age range, 30-67 years). In each case, inner retinal cleavage was located adjacent to retinal blood vessels. Tissue bridging the cleavage area was observed in ten eyes. Six eyes had epiretinal membranes (ERMs), two eyes had glaucoma, and one eye had ERM in addition to glaucoma. Six eyes with inner retinal cleavage without combined ocular abnormalities had highly myopic refractive error (-6.50 to -8.50 diopters). Cross-sectional OCT images of the areas of inner retinal cleavage demonstrated defects with irregular margins and empty spaces in the inner layers of the retina. During the follow-up period, no eye showed changes in inner retinal layer cleavage or visual field sensitivity. Inner retinal cleavage was found in eyes with high myopia or ERMs. Inner retinal cleavage was associated with structural changes distinct from those associated with glaucomatous RNFL defects.

  9. Invasive cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  10. Invasive cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    2002-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  11. Cleavage crystallography of liquid metal embrittled aluminum alloys

    NASA Technical Reports Server (NTRS)

    Reynolds, A. P.; Stoner, G. E.

    1991-01-01

    The crystallography of liquid metal-induced transgranular cleavage in six aluminum alloys having a variety of microstructures has been determined via Laue X-ray back reflection. The cleavage crystallography was independent of alloy microstructure, and the cleavage plane was 100-plane oriented in all cases. It was further determined that the cleavage crystallography was not influenced by alloy texture. Examination of the fracture surface indicated that there was not a unique direction of crack propagation. In addition, the existence of 100-plane cleavage on alloy 2024 fracture surfaces was inferred by comparison of secondary cleavage crack intersection geometry on the 2024 surfaces with the geometry of secondary cleavage crack intersections on the test alloys.

  12. Kinetics of hairpin ribozyme cleavage in yeast.

    PubMed Central

    Donahue, C P; Fedor, M J

    1997-01-01

    Hairpin ribozymes catalyze a self-cleavage reaction that provides a simple model for quantitative analyses of intracellular mechanisms of RNA catalysis. Decay rates of chimeric mRNAs containing self-cleaving ribozymes give a direct measure of intracellular cleavage kinetics in yeast. Intracellular ribozyme-mediated cleavage occurs at similar rates and shows similar inhibition by ribozyme mutations as ribozyme-mediated reactions in vitro, but only when ribozymes are located in a favorable mRNA sequence context. The impact of cleavage on mRNA abundance is shown to depend directly on intrinsic mRNA stability. Surprisingly, cleavage products are no more labile than uncleaved mRNAs despite the loss of terminal cap structures or poly (A). PMID:9292496

  13. Mechanisms for ribotoxin-induced ribosomal RNA cleavage

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    He, Kaiyu; Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824; Zhou, Hui-Ren

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥ 25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥ 10 ng/ml) and ribosome-inactivating protein ricin (≥ 300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activatedmore » kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. Highlights: ► Deoxynivalenol (DON) anisomycin, satratoxin G (SG) and ricin are ribotoxins. ► Ribotoxins induce 18s and 28s rRNA cleavage in the RAW 264.7 macrophage model. ► Ribotoxins induce r

  14. The invariant cleavage pattern displayed by ascidian embryos depends on spindle positioning along the cell's longest axis in the apical plane and relies on asynchronous cell divisions

    PubMed Central

    Dumollard, Rémi; Minc, Nicolas; Salez, Gregory; Aicha, Sameh Ben; Bekkouche, Faisal; Hebras, Céline; Besnardeau, Lydia; McDougall, Alex

    2017-01-01

    The ascidian embryo is an ideal system to investigate how cell position is determined during embryogenesis. Using 3D timelapse imaging and computational methods we analyzed the planar cell divisions in ascidian early embryos and found that spindles in every cell tend to align at metaphase in the long length of the apical surface except in cells undergoing unequal cleavage. Furthermore, the invariant and conserved cleavage pattern of ascidian embryos was found to consist in alternate planar cell divisions between ectoderm and endomesoderm. In order to test the importance of alternate cell divisions we manipulated zygotic transcription induced by β-catenin or downregulated wee1 activity, both of which abolish this cell cycle asynchrony. Crucially, abolishing cell cycle asynchrony consistently disrupted the spindle orienting mechanism underpinning the invariant cleavage pattern. Our results demonstrate how an evolutionary conserved cell cycle asynchrony maintains the invariant cleavage pattern driving morphogenesis of the ascidian blastula. DOI: http://dx.doi.org/10.7554/eLife.19290.001 PMID:28121291

  15. γ-Secretase Modulators and APH1 Isoforms Modulate γ-Secretase Cleavage but Not Position of ε-Cleavage of the Amyloid Precursor Protein (APP).

    PubMed

    Lessard, Christian B; Cottrell, Barbara A; Maruyama, Hiroko; Suresh, Suraj; Golde, Todd E; Koo, Edward H

    2015-01-01

    The relative increase in Aβ42 peptides from familial Alzheimer disease (FAD) linked APP and PSEN mutations can be related to changes in both ε-cleavage site utilization and subsequent step-wise cleavage. Cleavage at the ε-site releases the amyloid precursor protein (APP) intracellular domain (AICD), and perturbations in the position of ε-cleavage are closely associated with changes in the profile of amyloid β-protein (Aβ) species that are produced and secreted. The mechanisms by which γ-secretase modulators (GSMs) or FAD mutations affect the various γ-secretase cleavages to alter the generation of Aβ peptides have not been fully elucidated. Recent studies suggested that GSMs do not modulate ε-cleavage of APP, but the data were derived principally from recombinant truncated epitope tagged APP substrate. Here, using full length APP from transfected cells, we investigated whether GSMs modify the ε-cleavage of APP under more native conditions. Our results confirmed the previous findings that ε-cleavage is insensitive to GSMs. In addition, fenofibrate, an inverse GSM (iGSM), did not alter the position or kinetics of ε-cleavage position in vitro. APH1A and APH1B, a subunit of the γ-secretase complex, also modulated Aβ42/Aβ40 ratio without any alterations in ε-cleavage, a result in contrast to what has been observed with PS1 and APP FAD mutations. Consequently, GSMs and APH1 appear to modulate γ-secretase activity and Aβ42 generation by altering processivity but not ε-cleavage site utilization.

  16. Does Cleavage Work at Work? Men, but Not Women, Falsely Believe Cleavage Sells a Weak Product

    ERIC Educational Resources Information Center

    Glick, Peter; Chrislock, Karyna; Petersik, Korinne; Vijay, Madhuri; Turek, Aleksandra

    2008-01-01

    We examined whether men, but not women, would be distracted by a female sales representative's exposed cleavage, leading to greater perceived efficacy for a weak, but not for a strong product. A community sample of 88 men and 97 women viewed a video of a female pharmaceutical sales representative who (a) had exposed cleavage or dressed modestly…

  17. Experiments on schistosity and slaty cleavage

    USGS Publications Warehouse

    Becker, George Ferdinand

    1904-01-01

    Schistosity as a structure is important, and it is a part of the business of geologists to explain its origin. Slaty cleavage has further and greater importance as a possible tectonic feature. Scarcely a great mountain range exists, or has existed, along the course of which belts of slaty rock are not found, the dip of the cleavage usually approaching verticality. Are these slate belts equivalent to minutely distributed step faults of great total throw, or do they indicate compression perpendicular to the cleavage without attendant relative dislocation? Evidently the answer to this question is of first importance in the interpretation of orogenic phenomena.

  18. Intermolecular cleavage by UmuD-like mutagenesis proteins

    PubMed Central

    McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

    1998-01-01

    The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

  19. Enhancing Interleukin-6 and Interleukin-11 receptor cleavage.

    PubMed

    Lokau, Juliane; Wandel, Marieke; Garbers, Christoph

    2017-04-01

    Proteolytic cleavage of the membrane-bound Interleukin-6 receptor (IL-6R) by the metalloprotease ADAM17 releases an agonistic soluble form of the IL-6R (sIL-6R), which is responsible for the pro-inflammatory trans-signaling branch of the cytokine's activities. This proteolytic step, which is also called ectodomain shedding, is critically regulated by the cleavage site within the IL-6R stalk, because mutations or small deletions within this region are known to render the IL-6R irresponsive towards proteolysis. In the present study, we employed cleavage site profiling data of ADAM17 to generate an IL-6R with increased cleavage susceptibility. Using site-directed mutagenesis, we showed that the non-prime sites P3 and P2 and the prime site P1' were critical for this increase in proteolysis, whereas other positions within the cleavage site were of minor importance. Insertion of this optimized cleavage site into the stalk of the Interleukin-11 receptor (IL-11R) was not sufficient to enable ADAM17-mediated proteolysis, but transfer of different parts of the IL-6R stalk enabled shedding by ADAM17. These findings shed light on the cleavage site specificities of ADAM17 using a native substrate and reveal further differences in the proteolysis of IL-6R and IL-11R. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Specific Cleavage of the Nucleoprotein of Fish Rhabdovirus.

    PubMed

    Zhou, G-Z; Yi, Y-J; Chen, Z-Y; Zhang, Q-Y

    2015-11-01

    Siniperca chuatsi rhabdovirus (SCRV) is one of myriad rhabdoviruses recorded in fish. Preliminary data show that inhibition of the SCRV nucleoprotein (N) could significantly reduce the progeny virus titers in infected Epithelioma papulosum cyprinid (EPC) cells. Here, the authors propose that cleavage of the viral 47-kDa N protein is caspase-mediated based on caspase inhibition experiments, transient expression in EPC transfection, and analysis of cleavage sites. Cleavage of the SCRV N protein in culture was prevented by a pan-caspase inhibitor, z-VAD-FMK (z-Val-Ala-DL-Asp-fluoromethyl ketone). Subsequently, N was transiently expressed in EPC cells, the results of which indicated that the specific cleavage of N also occurred in the cells transfected with N-GFP plasmid. Several truncated fragments of the N gene were constructed and transiently transfected into EPC cells. Immunoblotting results indicated that D324 and D374 are the cleavage sites of N by caspases. The authors also found that z-VAD-FMK could inhibit the cytopathic effect in SCRV-infected EPC cells but not affect the production of infectious progeny, suggesting that the caspase-mediated cleavage of N protein is not required for in vitro SCRV replication. To the authors' knowledge, this is the first report on the cleavage of rhabdovirus proteins. © The Author(s) 2015.

  1. Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli.

    PubMed

    Hwang, Peter M; Pan, Jonathan S; Sykes, Brian D

    2014-01-21

    Today, proteins are typically overexpressed using solubility-enhancing fusion tags that allow for affinity chromatographic purification and subsequent removal by site-specific protease cleavage. In this review, we present an alternative approach to protein production using fusion partners specifically designed to accumulate in insoluble inclusion bodies. The strategy is appropriate for the mass production of short peptides, intrinsically disordered proteins, and proteins that can be efficiently refolded in vitro. There are many fusion protein systems now available for insoluble expression: TrpLE, ketosteroid isomerase, PurF, and PagP, for example. The ideal fusion partner is effective at directing a wide variety of target proteins into inclusion bodies, accumulates in large quantities in a highly pure form, and is readily solubilized and purified in commonly used denaturants. Fusion partner removal under denaturing conditions is biochemically challenging, requiring harsh conditions (e.g., cyanogen bromide in 70% formic acid) that can result in unwanted protein modifications. Recent advances in metal ion-catalyzed peptide bond cleavage allow for more mild conditions, and some methods involving nickel or palladium will likely soon appear in more biological applications. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  2. Quantification of DNA cleavage specificity in Hi-C experiments.

    PubMed

    Meluzzi, Dario; Arya, Gaurav

    2016-01-08

    Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Cleavage Entropy as Quantitative Measure of Protease Specificity

    PubMed Central

    Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

    2013-01-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

  4. New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

    PubMed Central

    Heo, Jinsol; Kim, Se Hyeuk

    2013-01-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13C-14, C-15C-15′, and C-13′C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro. PMID:23524669

  5. Verification of 2A peptide cleavage.

    PubMed

    Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

    2012-02-01

    The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. The easiest and most effective way to assess 2A cleavage is to perform transient transfection of 293T cells (human embryonic kidney cells) followed by western blot analysis, as described in this protocol. 293T cells are easy to grow and can be efficiently transfected with a variety of vectors. Cleavage can be assessed by detection with antibodies against the target proteins or anti-2A serum.

  6. Self-guide framing and persuasion: responsibly increasing message processing to ideal levels.

    PubMed

    Evans, Lisa M; Petty, Richard E

    2003-03-01

    The current research examines the effect that framing persuasive messages in terms of self-guides (ideal vs. ought) has on the attitudes and cognitive responses of individuals with chronic ideal versus ought self-guides. The strength of participants' ideal and ought self-guides and the magnitude of participants' ideal and ought self-discrepancies were measured using a computerized reaction time program. One week later, participants read a persuasive message about a fictional breakfast product, framed in terms of either ideals or oughts. Matching framing to stronger self-guide led to enhanced message processing activity, especially among individuals who were low in need for cognition. Individuals who read messages framed to match their stronger self-guides paid more attention to argument quality, as reflected in their attitudes and cognitive responses. Messages with self-guide framing that matched individuals' stronger self-discrepancies did not have this effect on processing.

  7. Functional analysis of coordinated cleavage in V(D)J recombination.

    PubMed

    Kim, D R; Oettinger, M A

    1998-08-01

    V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.

  8. Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

    PubMed Central

    Piao, Yi; Kimura, Ayano; Urano, Satomi; Saito, Yuhki; Taru, Hidenori; Yamamoto, Tohru; Hata, Saori; Suzuki, Toshiharu

    2013-01-01

    Background Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer's β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs. Methodology Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position. Conclusions The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer's disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by

  9. Microbial cleavage of organic C-S bonds

    DOEpatents

    Kilbane, II, John J.

    1994-01-01

    A microbial process for selective cleavage of organic C--S bonds which may be used for reducing the sulfur content of sulfur-containing organic carbonaceous materials, Microorganisms of Rhodococcus rhodochrous and Bacillus sphaericus have been found which have the ability of selective cleavage of organic C--S bonds. Particularly preferred microorganisms are Rhodococcus rhodochrous strain ATCC 53968 and Bacillus sphaericus strain ATCC 53969 and their derivatives.

  10. Characterization of Bleomycin-Mediated Cleavage of a Hairpin DNA Library

    PubMed Central

    Segerman, Zachary J.; Roy, Basab; Hecht, Sidney M.

    2013-01-01

    A study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of Fe(II)•BLM to effect cleavage on both the 3' and 5'-arms of the hairpin DNAs was characterized. The strongly bound DNAs were found to be efficient substrates for Fe•BLM A5-mediated hairpin DNA cleavage. Surprisingly, the most prevalent site of BLM-mediated cleavage was found to be the 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence, and other sequences generally not cleaved well by BLM when examined using arbitrarily chosen DNA substrates, were apparent when examining the library of ten hairpin DNAs. In total, 132 sites of DNA cleavage were produced by exposure of the hairpin DNA library to Fe•BLM A5. The existence of multiple sites of cleavage on both the 3′- and 5′-arms of the hairpin DNAs suggested that some of these might be double-strand cleavage events. Accordingly, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double-strand DNA damage. One hairpin DNA was characterized using this method, and gave results consistent with earlier reports of double-strand DNA cleavage, but with a sequence selectivity different from those reported previously. PMID:23834496

  11. Microbial cleavage of organic C-S bonds

    DOEpatents

    Kilbane, J.J. II.

    1994-10-25

    A microbial process is described for selective cleavage of organic C-S bonds which may be used for reducing the sulfur content of sulfur-containing organic carbonaceous materials. Microorganisms of Rhodococcus rhodochrous and Bacillus sphaericus have been found which have the ability of selective cleavage of organic C-S bonds. Particularly preferred microorganisms are Rhodococcus rhodochrous strain ATCC 53968 and Bacillus sphaericus strain ATCC 53969 and their derivatives.

  12. A Nanoindentation Study of the Plastic Deformation and Fracture Mechanisms in Single-Crystalline CaFe2As2

    NASA Astrophysics Data System (ADS)

    Frawley, Keara G.; Bakst, Ian; Sypek, John T.; Vijayan, Sriram; Weinberger, Christopher R.; Canfield, Paul C.; Aindow, Mark; Lee, Seok-Woo

    2018-04-01

    The plastic deformation and fracture mechanisms in single-crystalline CaFe2As2 has been studied using nanoindentation and density functional theory simulations. CaFe2As2 single crystals were grown in a Sn-flux, resulting in homogeneous and nearly defect-free crystals. Nanoindentation along the [001] direction produces strain bursts, radial cracking, and lateral cracking. Ideal cleavage simulations along the [001] and [100] directions using density functional theory calculations revealed that cleavage along the [001] direction requires a much lower stress than cleavage along the [100] direction. This strong anisotropy of cleavage strength implies that CaFe2As2 has an atomic-scale layered structure, which typically exhibits lateral cracking during nanoindentation. This special layered structure results from weak atomic bonding between the (001) Ca and Fe2As2 layers.

  13. Cleavage sites within the poliovirus capsid protein precursors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Larsen, G.R.; Anderson, C.W.; Dorner, A.J.

    1982-01-01

    Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occurmore » between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein.« less

  14. Binding and cleavage of nucleic acids by the "hairpin" ribozyme.

    PubMed

    Chowrira, B M; Burke, J M

    1991-09-03

    The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.

  15. Autotransporter structure reveals intra-barrel cleavage followed by conformational changes.

    PubMed

    Barnard, Travis J; Dautin, Nathalie; Lukacik, Petra; Bernstein, Harris D; Buchanan, Susan K

    2007-12-01

    Autotransporters are virulence factors produced by Gram-negative bacteria. They consist of two domains, an N-terminal 'passenger' domain and a C-terminal beta-domain. beta-domains form beta-barrel structures in the outer membrane while passenger domains are translocated into the extracellular space. In some autotransporters, the two domains are separated by proteolytic cleavage. Using X-ray crystallography, we solved the 2.7-A structure of the post-cleavage state of the beta-domain of EspP, an autotransporter produced by Escherichia coli strain O157:H7. The structure consists of a 12-stranded beta-barrel with the passenger domain-beta-domain cleavage junction located inside the barrel pore, approximately midway between the extracellular and periplasmic surfaces of the outer membrane. The structure reveals an unprecedented intra-barrel cleavage mechanism and suggests that two conformational changes occur in the beta-domain after cleavage, one conferring increased stability on the beta-domain and another restricting access to the barrel pore.

  16. Multi-Scale Effects in the Strength of Ceramics

    PubMed Central

    Cook, Robert F.

    2016-01-01

    Multiple length-scale effects are demonstrated in indentation-strength measurements of a range of ceramic materials under inert and reactive conditions. Meso-scale effects associated with flaw disruption by lateral cracking at large indentation loads are shown to increase strengths above the ideal indentation response. Micro-scale effects associated with toughening by microstructural restraints at small indentation loads are shown to decrease strengths below the ideal response. A combined meso-micro-scale analysis is developed that describes ceramic inert strength behaviors over the complete indentation flaw size range. Nano-scale effects associated with chemical equilibria and crack velocity thresholds are shown to lead to invariant minimum strengths at slow applied stressing rates under reactive conditions. A combined meso-micro-nano-scale analysis is developed that describes the full range of reactive and inert strength behaviors as a function of indentation load and applied stressing rate. Applications of the multi-scale analysis are demonstrated for materials design, materials selection, toughness determination, crack velocity determination, bond-rupture parameter determination, and prediction of reactive strengths. The measurements and analysis provide strong support for the existence of sharp crack tips in ceramics such that the nano-scale mechanisms of discrete bond rupture are separate from the larger scale crack driving force mechanics characterized by continuum-based stress-intensity factors. PMID:27563150

  17. Ideal cardiovascular health and its association with sedentary behaviour and fitness in psychiatric patients. The PsychiActive project.

    PubMed

    Bueno-Antequera, J; Oviedo-Caro, M Á; Munguía-Izquierdo, D

    2018-06-10

    Ideal cardiovascular health (CVH) was defined as meeting ideal levels of 4 health behaviours (smoking, body mass index, physical activity, and diet) and 3 biological factors (blood pressure, total cholesterol, and glucose) and is inversely related to cardiovascular disease and mortality. However, the prevalence of ideal CVH in patients with severe mental illness and the possible independent associations of sedentary behaviour and fitness with CVH score are unexplored. This study included 142 (34 women) outpatients with severe mental illness (primarily schizophrenia, n = 92). CVH was evaluated according to the American Heart Association guidelines. Sedentary behaviour, cardiorespiratory fitness, and muscular strength were measured by an activity-monitor, the 6-min walk test, and handgrip dynamometry. Cardiorespiratory fitness and strength values were combined in a composite fitness score. The prevalence of ideal CVH was: non-smoking (47.9%), body mass index (16.9), physical activity (83.1%), diet (10.4%), blood pressure (40.4%), total cholesterol (62.9%), and plasma glucose (66.7%). Low levels of sedentary behaviour and high cardiorespiratory, strength, and composite fitness score were associated with meeting the ideal threshold in most CVH metrics and having higher global CVH score; however, only cardiorespiratory and composite fitness score remained significantly related to global CVH score independent of sedentary behaviour and multiple confounders. Patients with severe mental illness generally have low prevalence of ideal CVH metrics, especially diet and body mass index. Additionally, our findings suggest the need or considering cardiorespiratory fitness, regardless of sedentary behaviour, to promote ideal CVH in this population. Copyright © 2018 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published

  18. Detection of nucleic acids by multiple sequential invasive cleavages

    DOEpatents

    Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  19. Detection of nucleic acids by multiple sequential invasive cleavages

    DOEpatents

    Hall, Jeff G; Lyamichev, Victor I; Mast, Andrea L; Brow, Mary Ann D

    2012-10-16

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  20. Not All Ideals are Equal: Intrinsic and Extrinsic Ideals in Relationships.

    PubMed

    Rodriguez, Lindsey M; Hadden, Benjamin W; Knee, C Raymond

    2015-03-01

    The ideal standards model suggests that greater consistency between ideal standards and actual perceptions of one's relationship predicts positive relationship evaluations; however, no research has evaluated whether this differs across types of ideals. A self-determination theory perspective was derived to test whether satisfaction of intrinsic ideals buffers the importance of extrinsic ideals. Participants (N=195) in committed relationships directly and indirectly reported the extent to which their partner met their ideal on two dimensions: intrinsic (e.g., warm, intimate) and extrinsic (e.g., attractive, successful). Relationship need fulfillment and relationship quality were also assessed. Hypotheses were largely supported, such that satisfaction of intrinsic ideals more strongly predicted relationship functioning, and satisfaction of intrinsic ideals buffered the relevance of extrinsic ideals for outcomes.

  1. Not All Ideals are Equal: Intrinsic and Extrinsic Ideals in Relationships

    PubMed Central

    Rodriguez, Lindsey M.; Hadden, Benjamin W.; Knee, C. Raymond

    2015-01-01

    The ideal standards model suggests that greater consistency between ideal standards and actual perceptions of one’s relationship predicts positive relationship evaluations; however, no research has evaluated whether this differs across types of ideals. A self-determination theory perspective was derived to test whether satisfaction of intrinsic ideals buffers the importance of extrinsic ideals. Participants (N=195) in committed relationships directly and indirectly reported the extent to which their partner met their ideal on two dimensions: intrinsic (e.g., warm, intimate) and extrinsic (e.g., attractive, successful). Relationship need fulfillment and relationship quality were also assessed. Hypotheses were largely supported, such that satisfaction of intrinsic ideals more strongly predicted relationship functioning, and satisfaction of intrinsic ideals buffered the relevance of extrinsic ideals for outcomes. PMID:25821396

  2. Detection of nucleic acid sequences by invader-directed cleavage

    DOEpatents

    Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

  3. Prediction of proprotein convertase cleavage sites.

    PubMed

    Duckert, Peter; Brunak, Søren; Blom, Nikolaj

    2004-01-01

    Many secretory proteins and peptides are synthesized as inactive precursors that in addition to signal peptide cleavage undergo post-translational processing to become biologically active polypeptides. Precursors are usually cleaved at sites composed of single or paired basic amino acid residues by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever has created additional focus on proprotein processing. We have developed a method for prediction of cleavage sites for PCs based on artificial neural networks. Two different types of neural networks have been constructed: a furin-specific network based on experimental results derived from the literature, and a general PC-specific network trained on data from the Swiss-Prot protein database. The method predicts cleavage sites in independent sequences with a sensitivity of 95% for the furin neural network and 62% for the general PC network. The ProP method is made publicly available at http://www.cbs.dtu.dk/services/ProP.

  4. Controllable laser thermal cleavage of sapphire wafers

    NASA Astrophysics Data System (ADS)

    Xu, Jiayu; Hu, Hong; Zhuang, Changhui; Ma, Guodong; Han, Junlong; Lei, Yulin

    2018-03-01

    Laser processing of substrates for light-emitting diodes (LEDs) offers advantages over other processing techniques and is therefore an active research area in both industrial and academic sectors. The processing of sapphire wafers is problematic because sapphire is a hard and brittle material. Semiconductor laser scribing processing suffers certain disadvantages that have yet to be overcome, thereby necessitating further investigation. In this work, a platform for controllable laser thermal cleavage was constructed. A sapphire LED wafer was modeled using the finite element method to simulate the thermal and stress distributions under different conditions. A guide groove cut by laser ablation before the cleavage process was observed to guide the crack extension and avoid deviation. The surface and cross section of sapphire wafers processed using controllable laser thermal cleavage were characterized by scanning electron microscopy and optical microscopy, and their morphology was compared to that of wafers processed using stealth dicing. The differences in luminous efficiency between substrates prepared using these two processing methods are explained.

  5. A Nanoindentation Study of the Plastic Deformation and Fracture Mechanisms in Single-Crystalline CaFe 2As 2

    DOE PAGES

    Frawley, Keara G.; Bakst, Ian; Sypek, John T.; ...

    2018-04-10

    In this paper, the plastic deformation and fracture mechanisms in single-crystalline CaFe 2As 2 has been studied using nanoindentation and density functional theory simulations. CaFe 2As 2 single crystals were grown in a Sn-flux, resulting in homogeneous and nearly defect-free crystals. Nanoindentation along the [001] direction produces strain bursts, radial cracking, and lateral cracking. Ideal cleavage simulations along the [001] and [100] directions using density functional theory calculations revealed that cleavage along the [001] direction requires a much lower stress than cleavage along the [100] direction. This strong anisotropy of cleavage strength implies that CaFe 2As 2 has an atomic-scalemore » layered structure, which typically exhibits lateral cracking during nanoindentation. This special layered structure results from weak atomic bonding between the (001) Ca and Fe 2As 2 layers.« less

  6. A Nanoindentation Study of the Plastic Deformation and Fracture Mechanisms in Single-Crystalline CaFe 2As 2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Frawley, Keara G.; Bakst, Ian; Sypek, John T.

    In this paper, the plastic deformation and fracture mechanisms in single-crystalline CaFe 2As 2 has been studied using nanoindentation and density functional theory simulations. CaFe 2As 2 single crystals were grown in a Sn-flux, resulting in homogeneous and nearly defect-free crystals. Nanoindentation along the [001] direction produces strain bursts, radial cracking, and lateral cracking. Ideal cleavage simulations along the [001] and [100] directions using density functional theory calculations revealed that cleavage along the [001] direction requires a much lower stress than cleavage along the [100] direction. This strong anisotropy of cleavage strength implies that CaFe 2As 2 has an atomic-scalemore » layered structure, which typically exhibits lateral cracking during nanoindentation. This special layered structure results from weak atomic bonding between the (001) Ca and Fe 2As 2 layers.« less

  7. GPS-CCD: A Novel Computational Program for the Prediction of Calpain Cleavage Sites

    PubMed Central

    Gao, Xinjiao; Ma, Qian; Ren, Jian; Xue, Yu

    2011-01-01

    As one of the most essential post-translational modifications (PTMs) of proteins, proteolysis, especially calpain-mediated cleavage, plays an important role in many biological processes, including cell death/apoptosis, cytoskeletal remodeling, and the cell cycle. Experimental identification of calpain targets with bona fide cleavage sites is fundamental for dissecting the molecular mechanisms and biological roles of calpain cleavage. In contrast to time-consuming and labor-intensive experimental approaches, computational prediction of calpain cleavage sites might more cheaply and readily provide useful information for further experimental investigation. In this work, we constructed a novel software package of GPS-CCD (Calpain Cleavage Detector) for the prediction of calpain cleavage sites, with an accuracy of 89.98%, sensitivity of 60.87% and specificity of 90.07%. With this software, we annotated potential calpain cleavage sites for hundreds of calpain substrates, for which the exact cleavage sites had not been previously determined. In this regard, GPS-CCD 1.0 is considered to be a useful tool for experimentalists. The online service and local packages of GPS-CCD 1.0 were implemented in JAVA and are freely available at: http://ccd.biocuckoo.org/. PMID:21533053

  8. IGF-1 receptor cleavage in hypertension.

    PubMed

    Cirrik, Selma; Schmid-Schönbein, Geert W

    2018-06-01

    Increased protease activity causes receptor dysfunction due to extracellular cleavage of different membrane receptors in hypertension. The vasodilatory effects of insulin-like growth factor-1 (IGF-1) are decreased in hypertension. Therefore, in the present study the association of an enhanced protease activity and IGF-1 receptor cleavage was investigated using the spontaneously hypertensive rats (SHRs) and their normotensive Wistar Kyoto (WKY) controls (n = 4). Matrix metalloproteinase (MMP) activities were determined using gelatin zymography on plasma and different tissue samples. WKY aorta rings were incubated in WKY or SHR plasma with or without MMP inhibitors, and immunohistochemistry was used to quantify the densities of the alpha and beta IGF-1 receptor (IGF-1R) subunits and to determine receptor cleavage. The pAkt and peNOS levels in the aorta were investigated using immunoblotting as a measure of IGF-IR function. Increased MMP-2 and MMP-9 activities were detected in plasma and peripheral tissues of SHRs. IGF-1R beta labeling was similar in both groups without plasma incubation, but the fraction of immunolabeled area for IGF-1R alpha was lower in the endothelial layer of the SHR aorta (p < 0.05). A 24-h incubation of WKY aorta with SHR plasma did not affect the IGF-1R beta labeling density, but reduced the IGF-1R alpha labeling density in the endothelium (p < 0.05). MMP inhibitors prevented this decrease (p < 0.01). Western blot analyses revealed that the pAkt and peNOS levels under IGF-1-stimulated and -unstimulated conditions were lower in SHRs (p < 0.05). A reduced IGF-1 cellular response in the aorta was associated with the decrease in the IGF-1R alpha subunit in the SHR hypertension model. Our results indicate that MMP-dependent receptor cleavage contributed to the reduced IGF-1 response in SHRs.

  9. Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

    PubMed

    Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

    1999-02-01

    We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

  10. Ab Initio energetics of SiO bond cleavage.

    PubMed

    Hühn, Carolin; Erlebach, Andreas; Mey, Dorothea; Wondraczek, Lothar; Sierka, Marek

    2017-10-15

    A multilevel approach that combines high-level ab initio quantum chemical methods applied to a molecular model of a single, strain-free SiOSi bridge has been used to derive accurate energetics for SiO bond cleavage. The calculated SiO bond dissociation energy and the activation energy for water-assisted SiO bond cleavage of 624 and 163 kJ mol -1 , respectively, are in excellent agreement with values derived recently from experimental data. In addition, the activation energy for H 2 O-assisted SiO bond cleavage is found virtually independent of the amount of water molecules in the vicinity of the reaction site. The estimated reaction energy for this process including zero-point vibrational contribution is in the range of -5 to 19 kJ mol -1 . © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  11. Sex Education and Ideals

    ERIC Educational Resources Information Center

    de Ruyter, Doret J.; Spiecker, Ben

    2008-01-01

    This article argues that sex education should include sexual ideals. Sexual ideals are divided into sexual ideals in the strict sense and sexual ideals in the broad sense. It is argued that ideals that refer to the context that is deemed to be most ideal for the gratification of sexual ideals in the strict sense are rightfully called sexual…

  12. Detection of nucleic acids by multiple sequential invasive cleavages 02

    DOEpatents

    Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

    2002-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  13. Cálculo del esfuerzo ideal de metales nobles mediante primeros principios en la dirección <100>

    NASA Astrophysics Data System (ADS)

    Bautista-Hernández, A.; López-Fuentes, M.; Pacheco-Espejel, V.; Rivas-Silva, J. F.

    2005-04-01

    We present calculations of the ideal strength on the < 100 > direction for noble metals (Cu, Ag and Au), by means of first principles calculations. First, we obtain the structural parameters (cell parameters, bulk modulus) for each studied metal. We deform on the < 100 > direction calculating the total energy and the stress tensor through the Hellman-Feynman theorem, by the relaxation of the unit cell in the perpendicular directions to the deformation one. The calculated cell constants differ 1.3 % from experimental data. The maximum ideal strength are 29.6, 17 and 19 GPa for Cu, Ag and Au respectively. Meanwhile, the calculated elastic modulus are 106 (Cu), 71 (Ag), and 45 GPa (Au) and are in agreement with the experimental values for polycrystalline samples. The values of maximum strength are explained by the optimum volume values due to the atomic radius size for each element.

  14. Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

    PubMed Central

    Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

    1999-01-01

    We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. PMID:9927419

  15. Development of a Novel Anti-HIV-1 Agent from within: Effect of Chimeric Vpr-Containing Protease Cleavage Site Residues on Virus Replication

    NASA Astrophysics Data System (ADS)

    Serio, D.; Rizvi, T. A.; Cartas, M.; Kalyanaraman, V. S.; Weber, I. T.; Koprowski, H.; Srinivasan, A.

    1997-04-01

    Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag-Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection.

  16. Efficient Cleavage of Ribosome-Associated Poly(A)-Binding Protein by Enterovirus 3C Protease

    PubMed Central

    Kuyumcu-Martinez, N. Muge; Joachims, Michelle; Lloyd, Richard E.

    2002-01-01

    Poliovirus (PV) causes a rapid and drastic inhibition of host cell cap-dependent protein synthesis during infection while preferentially allowing cap-independent translation of its own genomic RNA via an internal ribosome entry site element. Inhibition of cap-dependent translation is partly mediated by cleavage of an essential translation initiation factor, eIF4GI, during PV infection. In addition to cleavage of eIF4GI, cleavage of eIF4GII and poly(A)-binding protein (PABP) has been recently proposed to contribute to complete host translation shutoff; however, the relative importance of eIF4GII and PABP cleavage has not been determined. At times when cap-dependent translation is first blocked during infection, only 25 to 35% of the total cellular PABP is cleaved; therefore, we hypothesized that the pool of PABP associated with polysomes may be preferentially targeted by viral proteases. We have investigated what cleavage products of PABP are produced in vivo and the substrate determinants for cleavage of PABP by 2A protease (2Apro) or 3C protease (3Cpro). Our results show that PABP in ribosome-enriched fractions is preferentially cleaved in vitro and in vivo compared to PABP in other fractions. Furthermore, we have identified four N-terminal PABP cleavage products produced during PV infection and have shown that viral 3C protease generates three of the four cleavage products. Also, 3Cpro is more efficient in cleaving PABP in ribosome-enriched fractions than 2Apro in vitro. In addition, binding of PABP to poly(A) RNA stimulates 3Cpro-mediated cleavage and inhibits 2Apro-mediated cleavage. These results suggest that 3Cpro plays a major role in processing PABP during virus infection and that the interaction of PABP with translation initiation factors, ribosomes, or poly(A) RNA may promote its cleavage by viral 2A and 3C proteases. PMID:11836384

  17. Protein cleavage strategies for an improved analysis of the membrane proteome

    PubMed Central

    Fischer, Frank; Poetsch, Ansgar

    2006-01-01

    Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms. PMID:16512920

  18. Cleavage in conical sand dollar eggs.

    PubMed

    Rappaport, R; Rappaport, B N

    1994-07-01

    Previous experiments have shown that the mitotic apparatus and the surface can interact and produce functional furrows in various unusual geometrical circumstances. The consistent development of the furrow in the plane equidistant from the aster centers has led to conjecture about the need for a special structural configuration of the subsurface in the future cleavage plane. In most experiments involving altered cell geometry, the relation between each aster and nearby surface was symmetrical, and the effect of that symmetry upon the position and orientation of the cleavage mechanism in the cortex has not been systematically analyzed. The normal symmetry of sand dollar eggs can be changed by reshaping them into cones. When the cone and mitotic axes are parallel, the aster center closer to the vertex is also closer to the nearby surface, and the cleavage plane develops on the vertex side of the midpoint between the asters. A mitotic apparatus oriented perpendicular to the cone axis produces in the base of the cone a normal unilateral furrow that advances toward the vertex, and a second contractile band that isolates the vertex region. This event only occurs when the surface is conical and the mitotic apparatus is perpendicular to the cone axis. Furrow formation is not restricted to the plane of the metaphase plate or the midpoint between the aster centers. The orientation of mitotic apparatus-produced contractile bands is not limited to the circumstances in normal cytokinesis, but may vary according to surface contour. These results confirm predictions of the Harris and Gewalt model of contractile ring induction.

  19. Flanking signal and mature peptide residues influence signal peptide cleavage

    PubMed Central

    Choo, Khar Heng; Ranganathan, Shoba

    2008-01-01

    Background Signal peptides (SPs) mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I), and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i) eukaryotes (Euk) (ii) Gram-positive (Gram+) bacteria, and (iii) Gram-negative (Gram-) bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs. PMID:19091014

  20. Efficient plasmid DNA cleavage by a mononuclear copper(II) complex.

    PubMed

    Sissi, Claudia; Mancin, Fabrizio; Gatos, Maddalena; Palumbo, Manlio; Tecilla, Paolo; Tonellato, Umberto

    2005-04-04

    The Cu(II) complex of the ligand all-cis-2,4,6-triamino-1,3,5-trihydroxycyclohexane (TACI) is a very efficient catalyst of the cleavage of plasmid DNA in the absence of any added cofactor. The maximum rate of degradation of the supercoiled plasmid DNA form, obtained at pH 8.1 and 37 degrees C, in the presence of 48 microM TACI.Cu(II), is 2.3 x 10(-3) s(-1), corresponding to a half-life time of only 5 min for the cleavage of form I (supercoiled) to form II (relaxed circular). The dependence of the rate of plasmid DNA cleavage from the TACI.Cu(II) complex concentration follows an unusual and very narrow bell-like profile, which suggests an high DNA affinity of the complexes but also a great tendency to form unreactive dimers. The reactivity of the TACI.Cu(II) complexes is not affected by the presence of several scavengers for reactive oxygen species or when measured under anaerobic conditions. Moreover, no degradation of the radical reporter Rhodamine B is observed in the presence of such complexes. These results are consistent with the operation of a prevailing hydrolytic pathway under the normal conditions used, although the failure to obtain enzymatic religation of the linearized DNA does not allow one to rule out the occurrence of a nonhydrolytic oxygen-independent cleavage. A concurrent oxidative mechanism becomes competitive upon addition of reductants or in the presence of high levels of molecular oxygen: under such conditions, in fact, a remarkable increase in the rate of DNA cleavage is observed.

  1. Site-Specific Pyrolysis Induced Cleavage at Aspartic Acid Residue in Peptides and Proteins

    PubMed Central

    Zhang, Shaofeng; Basile, Franco

    2011-01-01

    A simple and site-specific non-enzymatic method based on pyrolysis has been developed to cleave peptides and proteins. Pyrolytic cleavage was found to be specific and rapid as it induced a cleavage at the C-terminal side of aspartic acid in the temperature range of 220–250 °C in 10 seconds. Electrospray Ionization (ESI) mass spectrometry (MS) and tandem-MS (MS/MS) were used to characterize and identify pyrolysis cleavage products, confirming that sequence information is conserved after the pyrolysis process in both peptides and protein tested. This suggests that pyrolysis-induced cleavage at aspartyl residues can be used as a rapid protein digestion procedure for the generation of sequence specific protein biomarkers. PMID:17388620

  2. Selection of hammerhead ribozymes for optimum cleavage of interleukin 6 mRNA.

    PubMed Central

    Hendrix, C; Anné, J; Joris, B; Van Aerschot, A; Herdewijn, P

    1996-01-01

    Four GUC triplets in the coding region of the MRNA of interleukin 6 (IL-6) were examined for their suitabilty to serve as a target for hammerhead ribozome-mediated cleavage. This selection procedure was performed with the intention to downregulate IL-6 production as a potential treatment of those diseases in which IL-6 overexpression is involved. Hammerhead ribozymes and their respective short synthetic substrates (19-mers) were synthesized for these four GUC triplets. Notwithstanding the identical catalytic core sequences, the difference in base composition of the helices involved in substrate binding caused substantial variation in cleavage activity. The cleavage reactions on the 1035 nucleotide IL-6 mRNA transcript revealed that two ribozymes were able to cleave this substrate, showing a decrease in catalytic efficiency to 1/30 and 1/300 of the short substrate. This study indicates that the GUC triplet located at nucleotide 510 of the mRNA of IL-6 is the best site for hammerhead ribozyme-mediated cleavage. We suggest that in future targeting of chemically modified hammerhead ribosomes for cleavage of IL-6 RNA should be directed at this location. PMID:8670082

  3. Measurement of optical Feshbach resonances in an ideal gas.

    PubMed

    Blatt, S; Nicholson, T L; Bloom, B J; Williams, J R; Thomsen, J W; Julienne, P S; Ye, J

    2011-08-12

    Using a narrow intercombination line in alkaline earth atoms to mitigate large inelastic losses, we explore the optical Feshbach resonance effect in an ultracold gas of bosonic (88)Sr. A systematic measurement of three resonances allows precise determinations of the optical Feshbach resonance strength and scaling law, in agreement with coupled-channel theory. Resonant enhancement of the complex scattering length leads to thermalization mediated by elastic and inelastic collisions in an otherwise ideal gas. Optical Feshbach resonance could be used to control atomic interactions with high spatial and temporal resolution.

  4. Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA.

    PubMed

    Dayeh, Daniel M; Cantara, William A; Kitzrow, Jonathan P; Musier-Forsyth, Karin; Nakanishi, Kotaro

    2018-06-12

    The recent identification and development of RNA-guided enzymes for programmable cleavage of target nucleic acids offers exciting possibilities for both therapeutic and biotechnological applications. However, critical challenges such as expensive guide RNAs and inability to predict the efficiency of target recognition, especially for highly-structured RNAs, remain to be addressed. Here, we introduce a programmable RNA restriction enzyme, based on a budding yeast Argonaute (AGO), programmed with cost-effective 23-nucleotide (nt) single-stranded DNAs as guides. DNA guides offer the advantage that diverse sequences can be easily designed and purchased, enabling high-throughput screening to identify optimal recognition sites in the target RNA. Using this DNA-induced slicing complex (DISC) programmed with 11 different guide DNAs designed to span the sequence, sites of cleavage were identified in the 352-nt human immunodeficiency virus type 1 5'-untranslated region. This assay, coupled with primer extension and capillary electrophoresis, allows detection and relative quantification of all DISC-cleavage sites simultaneously in a single reaction. Comparison between DISC cleavage and RNase H cleavage reveals that DISC not only cleaves solvent-exposed sites, but also sites that become more accessible upon DISC binding. This study demonstrates the advantages of the DISC system for programmable cleavage of highly-structured, functional RNAs.

  5. Ideals versus reality: Are weight ideals associated with weight change in the population?

    PubMed

    Kärkkäinen, Ulla; Mustelin, Linda; Raevuori, Anu; Kaprio, Jaakko; Keski-Rahkonen, Anna

    2016-04-01

    To quantify weight ideals of young adults and to examine whether the discrepancy between actual and ideal weight is associated with 10-year body mass index (BMI) change in the population. This study comprised 4,964 adults from the prospective population-based FinnTwin16 study. They reported their actual and ideal body weight at age 24 (range 22-27) and 10 years later (attrition 24.6%). The correlates of discrepancy between actual and ideal body weight and the impact on subsequent BMI change were examined. The discrepancy between actual and ideal weight at 24 years was on average 3.9 kg (1.4 kg/m(2) ) among women and 1.2 kg (0.4 kg/m(2) ) among men. On average, participants gained weight during follow-up irrespective of baseline ideal weight: women ¯x = +4.8 kg (1.7 kg/m(2) , 95% CI 1.6-1.9 kg/m(2) ), men ¯x = +6.3 kg (2.0 kg/m(2) , 95% CI 1.8-2.1 kg/m(2) ). Weight ideals at 24 years were not correlated with 10-year weight change. At 34 years, just 13.2% of women and 18.9% of men were at or below the weight they had specified as their ideal weight at 24 years. Women and men adjusted their ideal weight upward over time. Irrespective of ideal weight at baseline, weight gain was nearly universal. Weight ideals were shifted upward over time. © 2016 The Obesity Society.

  6. Reactions involving the heterolytic cleavage of carbon-element σ-bonds by Grignard reagents

    NASA Astrophysics Data System (ADS)

    Polivin, Yurii N.; Karakhanov, Robert A.; Postnov, Victor N.

    1990-03-01

    The reactions involving the heterolysis of the C-O, C-C, C-N, C-S, C-Cl, etc. bonds by organomagnesium compounds are examined and the nature of this interesting phenomenon is analysed. On the basis of the analysis of the characteristic features of the cleavage under discussion, it is shown that the heterolysis of the carbon-element bond is, firstly, a general reaction for all classes of organic compounds (provided that two conditions are observed: the substrate molecule must fragment into two stable species — a carbonium ion and an anion — and the strength of the Lewis acid properties should be adequate for the occurrence of the above reaction) and, secondly, the heterolysis of the carbon-element bond is one of the independent pathways in the reactions of the Grignard reagents. The bibliography includes 158 references.

  7. Review of Idealized Aircraft Wake Vortex Models

    NASA Technical Reports Server (NTRS)

    Ahmad, Nashat N.; Proctor, Fred H.; Duparcmeur, Fanny M. Limon; Jacob, Don

    2014-01-01

    Properties of three aircraft wake vortex models, Lamb-Oseen, Burnham-Hallock, and Proctor are reviewed. These idealized models are often used to initialize the aircraft wake vortex pair in large eddy simulations and in wake encounter hazard models, as well as to define matched filters for processing lidar observations of aircraft wake vortices. Basic parameters for each vortex model, such as peak tangential velocity and circulation strength as a function of vortex core radius size, are examined. The models are also compared using different vortex characterizations, such as the vorticity magnitude. Results of Euler and large eddy simulations are presented. The application of vortex models in the postprocessing of lidar observations is discussed.

  8. (Fuzzy) Ideals of BN-Algebras

    PubMed Central

    Walendziak, Andrzej

    2015-01-01

    The notions of an ideal and a fuzzy ideal in BN-algebras are introduced. The properties and characterizations of them are investigated. The concepts of normal ideals and normal congruences of a BN-algebra are also studied, the properties of them are displayed, and a one-to-one correspondence between them is presented. Conditions for a fuzzy set to be a fuzzy ideal are given. The relationships between ideals and fuzzy ideals of a BN-algebra are established. The homomorphic properties of fuzzy ideals of a BN-algebra are provided. Finally, characterizations of Noetherian BN-algebras and Artinian BN-algebras via fuzzy ideals are obtained. PMID:26125050

  9. A matrix solution for the simulation of magnetic fields with ideal current loops

    NASA Technical Reports Server (NTRS)

    Stankiewicz, N.

    1979-01-01

    A matrix formulation is presented for describing axisymmetric magnetic field data with ideal current loops. A computer program written in APL is used to invert the matrix and hence to solve for the coil strengths which are used to represent the field data. Examples are given of the coil representation for (1) measured magnetic data, (2) refocusing fields, and (3) PPM focusing fields.

  10. Domainal cleavage as an Anisotropic Reaction-diffusion Process

    NASA Astrophysics Data System (ADS)

    Mulchrone, Kieran; Meere, Patrick

    2017-04-01

    Domainal cleavage comprises zones dominated by quartz and feldspar (QF-domains) and zones dominated by Mica (M-domains) which form at low metamorphic grades. The protolith is typically fairly homogeneous mudstone, siltstone, sandstone or limestone. Wet diffusion or pressure solution along grain boundaries is a key mechanism in the development of domanial cleavage. However, this does not explain why M-domains become sub-regularly spaced, visually evident in coarser-grained rocks, and take on an anastomising morphology. The ratio of M to QF-domains by volume can range from 1 to 0.1 and lower i.e. in extreme cases M-domains are intermittent but regularly spaced. It is suggested here that an anisotropic reaction-diffusion process model can explain these features. The imposed stress field instantaneously leads to anisotropy of diffusion by narrowing intergranular channels perpendicular to the principal stress. This leads to a preferred diffusion of chemicals parallel to the principal stress direction and lower diffusion rates in the normal direction. Combining this with the chemical reaction of pressure solution produces an anisotropic reaction-diffusion system. Both isotropic and anistropic reaction diffusion systems lead to pattern formation as discovered by Alan Turing on the 1950's as an explanation for patterns found in animal skins such as spots and stripes. Thus domanial cleavage is a striped pattern induced by diffusion anisotropy combined with a chemical reaction. Furthermore, rates of chemical reaction in intergranular fluids is likely to be many orders of magnitude greater that rates of deformation. Therefore we expect domanial cleavage to form relatively rapidly. As deformation progresses the M-domains behave less competently and may be the site of enhanced shearing. An example from Co. Cork, Ireland demonstrates shear folding in low-grade metasedimentary rocks with reverse shear along M-domains at a high angle to the maximum compressive stress.

  11. Changes in Contact Area in Meniscus Horizontal Cleavage Tears Subjected to Repair and Resection.

    PubMed

    Beamer, Brandon S; Walley, Kempland C; Okajima, Stephen; Manoukian, Ohan S; Perez-Viloria, Miguel; DeAngelis, Joseph P; Ramappa, Arun J; Nazarian, Ara

    2017-03-01

    To assess the changes in tibiofemoral contact pressure and contact area in human knees with a horizontal cleavage tear before and after treatment. Ten human cadaveric knees were tested. Pressure sensors were placed under the medial meniscus and the knees were loaded at twice the body weight for 20 cycles at 0°, 10°, and 20° of flexion. Contact area and pressure were recorded for the intact meniscus, the meniscus with a horizontal cleavage tear, after meniscal repair, after partial meniscectomy (single leaflet), and after subtotal meniscectomy (double leaflet). The presence of a horizontal cleavage tear significantly increased average peak contact pressure and reduced effective average tibiofemoral contact area at all flexion angles tested compared with the intact state (P < .03). There was approximately a 70% increase in contact pressure after creation of the horizontal cleavage tear. Repairing the horizontal cleavage tear restored peak contact pressures and areas to within 15% of baseline, statistically similar to the intact state at all angles tested (P < .05). Partial meniscectomy and subtotal meniscectomy significantly increased average peak contact pressure and reduced average contact area at all degrees of flexion compared with the intact state (P < .05). The presence of a horizontal cleavage tear in the medial meniscus causes a significant reduction in contact area and a significant elevation in contact pressure. These changes may accelerate joint degeneration. A suture-based repair of these horizontal cleavage tears returns the contact area and contact pressure to nearly normal, whereas both partial and subtotal meniscectomy lead to significant reductions in contact area and significant elevations in contact pressure within the knee. Repairing horizontal cleavage tears may lead to improved clinical outcomes by preserving meniscal tissue and the meniscal function. Understanding contact area and peak contact pressure resulting from differing strategies

  12. Variable context Markov chains for HIV protease cleavage site prediction.

    PubMed

    Oğul, Hasan

    2009-06-01

    Deciphering the knowledge of HIV protease specificity and developing computational tools for detecting its cleavage sites in protein polypeptide chain are very desirable for designing efficient and specific chemical inhibitors to prevent acquired immunodeficiency syndrome. In this study, we developed a generative model based on a generalization of variable order Markov chains (VOMC) for peptide sequences and adapted the model for prediction of their cleavability by certain proteases. The new method, called variable context Markov chains (VCMC), attempts to identify the context equivalence based on the evolutionary similarities between individual amino acids. It was applied for HIV-1 protease cleavage site prediction problem and shown to outperform existing methods in terms of prediction accuracy on a common dataset. In general, the method is a promising tool for prediction of cleavage sites of all proteases and encouraged to be used for any kind of peptide classification problem as well.

  13. Oxidative cleavage of erucic acid for the synthesis of brassylic acid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mohammed J. Nasrullah; Pooja Thapliyal; Erica N. Pfarr

    2010-10-29

    The main focus of this work is to synthesize Brassylic Acid (BA) using oxidative cleavage of Erucic Acid (EA). Crambe (Crambe abyssinica) is an industrial oilseed grown in North Dakota. Crambe has potential as an industrial fatty acid feedstock as a source of Erucic acid (EA). It has approximately 50-60 % of EA, a C{sub 22} monounsaturated fatty acid. Oxidative cleavage of unsaturated fatty acids derived from oilseeds produces long chain (9, 11, and 13 carbon atoms) dibasic and monobasic acids. These acids are known commercial feedstocks for the preparation of nylons, polyesters, waxes, surfactants, and perfumes. Other sources ofmore » EA are Rapeseed seed oil which 50-60 % of EA. Rapeseed is grown outside USA. The oxidative cleavage of EA was done using a high throughput parallel pressure reactor system. Kinetics of the reaction shows that BA yields reach a saturation at 12 hours. H{sub 2}WO{sub 4} was found to be the best catalyst for the oxidative cleavage of EA. High yields of BA were obtained at 80 C with bubbling of O{sub 2} or 10 bar of O{sub 2} for 12 hours.« less

  14. Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly.

    PubMed Central

    Ryner, L C; Takagaki, Y; Manley, J L

    1989-01-01

    To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively. Images PMID:2566911

  15. Phosphorylation-dependent cleavage regulates von Hippel Lindau proteostasis and function

    PubMed Central

    German, Peter; Bai, Shanshan; Liu, Xian-De; Sun, Mianen; Zhou, Lijun; Kalra, Sarathi; Zhang, Xuesong; Minelli, Rosalba; Scott, Kenneth L.; Mills, Gordon B.; Jonasch, Eric; Ding, Zhiyong

    2016-01-01

    Loss of von Hippel Lindau (VHL) protein function is a key driver of VHL diseases, including sporadic and inherited clear cell renal cell carcinoma. Modulation of the proteostasis of VHL, especially missense point-mutated VHL, is a promising approach to augmenting VHL levels and function. VHL proteostasis is regulated by multiple mechanisms including folding, chaperone binding, complex formation, and phosphorylation. Nevertheless, many details underlying the regulations of VHL proteostasis are unknown. VHL is expressed as two variants, VHL30 and VHL19. Furthermore, the long form variant of VHL was often detected as multiple bands by Western blotting. However, how these multiple species of VHL are generated and whether the process regulates VHL proteostasis and function are unknown. We hypothesized that the two major species are generated by VHL protein cleavage, and the cleavage regulates VHL proteostasis and subsequent function. We characterized VHL species using genetic and pharmacologic approaches and showed that VHL was first cleaved at the N-terminus by chymotrypsin C before being directed for proteasomal degradation. Casein kinase 2-mediated phosphorylation at VHL N-terminus was required for the cleavage. Furthermore, inhibition of cleavage stabilized VHL protein, thereby promoting HIF downregulation. Our study reveals a novel mechanism regulating VHL proteostasis and function, which is significant for identifying new drug targets and developing new therapeutic approaches targeting VHL deficiency in VHL diseases. PMID:26973240

  16. Specificity of hammerhead ribozyme cleavage.

    PubMed Central

    Hertel, K J; Herschlag, D; Uhlenbeck, O C

    1996-01-01

    To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths. Images PMID:8670879

  17. Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

    PubMed Central

    Borda, Emily J.; Markley, John C.; Sigurdsson, Snorri Th.

    2003-01-01

    We have characterized a novel Zn2+-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn2+. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn2+-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes. PMID:12736309

  18. Ideal discrimination of discrete clinical endpoints using multilocus genotypes.

    PubMed

    Hahn, Lance W; Moore, Jason H

    2004-01-01

    Multifactor Dimensionality Reduction (MDR) is a method for the classification and prediction of discrete clinical endpoints using attributes constructed from multilocus genotype data. Empirical studies with both real and simulated data suggest that MDR has good power for detecting gene-gene interactions in the absence of independent main effects. The purpose of this study is to develop an objective, theory-driven approach to evaluate the strengths and limitations of MDR. To accomplish this goal, we borrow concepts from ideal observer analysis used in visual perception to evaluate the theoretical limits of classifying and predicting discrete clinical endpoints using multilocus genotype data. We conclude that MDR ideally discriminates between low risk and high risk subjects using attributes constructed from multilocus genotype data. We also how that the classification approach used once a multilocus attribute is constructed is similar to that of a naive Bayes classifier. This study provides a theoretical foundation for the continued development, evaluation, and application of the MDR as a data mining tool in the domain of statistical genetics and genetic epidemiology.

  19. Ideal glass transitions in thin films: An energy landscape perspective

    NASA Astrophysics Data System (ADS)

    Truskett, Thomas M.; Ganesan, Venkat

    2003-07-01

    We introduce a mean-field model for the potential energy landscape of a thin fluid film confined between parallel substrates. The model predicts how the number of accessible basins on the energy landscape and, consequently, the film's ideal glass transition temperature depend on bulk pressure, film thickness, and the strength of the fluid-fluid and fluid-substrate interactions. The predictions are in qualitative agreement with the experimental trends for the kinetic glass transition temperature of thin films, suggesting the utility of landscape-based approaches for studying the behavior of confined fluids.

  20. Pressure modulates the self-cleavage step of the hairpin ribozyme

    NASA Astrophysics Data System (ADS)

    Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

    2017-03-01

    The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

  1. Reductive cleavage of the peptide bond

    NASA Technical Reports Server (NTRS)

    Holian, J.; Garrison, W. M.

    1973-01-01

    In many biological research efforts, long chain organic molecules are studied by breaking large molecules into smaller components. Cleavage technique of recent interest is the use of solvated electrons. These are formed when aqueous solutions are bombarded with gamma radiation. Solvated electron is very reactive and can reduce most any species present, even to form free radicals.

  2. SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3' end processing.

    PubMed

    Yu, Simei; Jordán-Pla, Antonio; Gañez-Zapater, Antoni; Jain, Shruti; Rolicka, Anna; Östlund Farrants, Ann-Kristin; Visa, Neus

    2018-05-31

    SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3' end maturation by facilitating 3' end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3' end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.

  3. Stille coupling via C-N bond cleavage

    NASA Astrophysics Data System (ADS)

    Wang, Dong-Yu; Kawahata, Masatoshi; Yang, Ze-Kun; Miyamoto, Kazunori; Komagawa, Shinsuke; Yamaguchi, Kentaro; Wang, Chao; Uchiyama, Masanobu

    2016-09-01

    Cross-coupling is a fundamental reaction in the synthesis of functional molecules, and has been widely applied, for example, to phenols, anilines, alcohols, amines and their derivatives. Here we report the Ni-catalysed Stille cross-coupling reaction of quaternary ammonium salts via C-N bond cleavage. Aryl/alkyl-trimethylammonium salts [Ar/R-NMe3]+ react smoothly with arylstannanes in 1:1 molar ratio in the presence of a catalytic amount of commercially available Ni(cod)2 and imidazole ligand together with 3.0 equivalents of CsF, affording the corresponding biaryl with broad functional group compatibility. The reaction pathway, including C-N bond cleavage step, is proposed based on the experimental and computational findings, as well as isolation and single-crystal X-ray diffraction analysis of Ni-containing intermediates. This reaction should be widely applicable for transformation of amines/quaternary ammonium salts into multi-aromatics.

  4. N-CADHERIN PRODOMAIN CLEAVAGE REGULATES SYNAPSE FORMATION IN VIVO

    PubMed Central

    Latefi, Nazlie S.; Pedraza, Liliana; Schohl, Anne; Li, Ziwei; Ruthazer, Edward S.

    2009-01-01

    Cadherins are initially synthesized bearing a prodomain that is thought to limit adhesion during early stages of biosynthesis. Functional cadherins lack this prodomain, raising the intriguing possibility that cells may utilize prodomain cleavage as a means to temporally or spatially regulate adhesion after delivery of cadherin to the cell surface. In support of this idea, immunostaining for the prodomain of zebrafish N-cadherin revealed enriched labeling at neuronal surfaces at the soma and along axonal processes. To determine whether post-translational cleavage of the prodomain affects synapse formation, we imaged Rohon-Beard cells in zebrafish embryos expressing GFP-tagged wild-type N-cadherin (NCAD-GFP) or a GFP-tagged N-cadherin mutant expressing an uncleavable prodomain (PRON-GFP) rendering it non-adhesive. NCAD-GFP accumulated at synaptic microdomains in a developmentally regulated manner, and its overexpression transiently accelerated synapse formation. PRON-GFP was much more diffusely distributed along the axon and its overexpression delayed synapse formation. Our results support the notion that N-cadherin serves to stabilize pre- to postsynaptic contacts early in synapse development and suggests that regulated cleavage of the N-cadherin prodomain may be a mechanism by which the kinetics of synaptogenesis are regulated. PMID:19365814

  5. Real-time single cell analysis of Bid cleavage and translocation in cisplatin-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Xing, Da; Pei, Yihui; Chen, Wei R.

    2007-02-01

    Cancer cell apoptosis can be induced by cisplatin, an efficient anticancer agent. However, its mechanism is not fully understood. Bcl-2 homology domain (BH) 3-only proteins couple stress signals to mitochondrial apoptotic pathways. Calpain-mediated cleavage of the BH3-only protein Bid into a 14 kD truncated protein (tBid) has been implicated in cisplatin-induced apoptotic pathway. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage during cisplatin-induced apoptosis in ASTC-a-1 cells. The cells were also co-transfected with Bid-CFP and DsRed-Mit to dynamically detect tBid translocation. Cells showed a cleavage of the Bid-FRET probe occurring at about 4-5 h after treated with 20 µM cisplatin. Cleavage of the Bid-FRET probe coincided with a translocation of tBid from the cytosolic to the mitochondria, and the translocation lasted about 1.5 h. Using real-time single-cell analysis, we first observed the kinetics of Bid cleavage and translocation to mitochondria in living cells during cisplatin-induced apoptosis.

  6. Bond cleavage of lignin model compounds into aromatic monomers using supported metal catalysts in supercritical water

    PubMed Central

    Yamaguchi, Aritomo; Mimura, Naoki; Shirai, Masayuki; Sato, Osamu

    2017-01-01

    More efficient use of lignin carbon is necessary for carbon-efficient utilization of lignocellulosic biomass. Conversion of lignin into valuable aromatic compounds requires the cleavage of C–O ether bonds and C–C bonds between lignin monomer units. The catalytic cleavage of C–O bonds is still challenging, and cleavage of C–C bonds is even more difficult. Here, we report cleavage of the aromatic C–O bonds in lignin model compounds using supported metal catalysts in supercritical water without adding hydrogen gas and without causing hydrogenation of the aromatic rings. The cleavage of the C–C bond in bibenzyl was also achieved with Rh/C as a catalyst. Use of this technique may greatly facilitate the conversion of lignin into valuable aromatic compounds. PMID:28387304

  7. The Place of Ideals in Teaching.

    ERIC Educational Resources Information Center

    Hansen, David T.

    This paper examines whether ideals and idealism have a role to play in teaching, identifying some ambiguities and problems associated with ideals and arguing that ideals figure importantly in teaching, but they are ideals of character or personhood as much as they are ideals of educational purpose. The first section focuses on the promise and…

  8. Experimental and Theoretical Investigations on Bond Strength of GFRP Rebars in Normal and High Strength Concrete

    NASA Astrophysics Data System (ADS)

    Eswanth, P.; Dhinakaran, G.

    2017-07-01

    Bond behavior between GFRP bars and concrete is the most important parameter for constructing corrosion free structures by implementing the material. Serviceability of reinforced concrete structures are controlled by bond behavior. GFRP materials behave differently from reinforcing steel in terms of bond. They are of non-homogeneous and anisotropic. Due to this outstanding behavior, there is a difference in transfer of loads between GFRP bars and concrete which made it as an idealized choice of a material. In the present work, the bond strength of GFRP bars in normal and high strength concrete was studied. In total, 12 specimens containing 12 mm, 16 mm diameter rebars which were embedded in 150 mm x 150 mm x 150 mm cubes were investigated. The specimens were subjected to direct tension pull out test in accordance with IS 2770 part 1. The comparison of bond properties of GFRP rebar in normal and high strength concrete showed that pull out load of non-metallic rebar fell well within the range.

  9. Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

    PubMed

    Mekkat, Arya; Poppleton, Erik; An, Bo; Visse, Robert; Nagase, Hideaki; Kaplan, David L; Brodsky, Barbara; Lin, Yu-Shan

    2018-05-12

    Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage. Copyright © 2018. Published by Elsevier Inc.

  10. Medical ethics and more: ideal theories, non-ideal theories and conscientious objection.

    PubMed

    Luna, Florencia

    2015-01-01

    Doing 'good medical ethics' requires acknowledgment that it is often practised in non-ideal circumstances! In this article I present the distinction between ideal theory (IT) and non-ideal theory (NIT). I show how IT may not be the best solution to tackle problems in non-ideal contexts. I sketch a NIT framework as a useful tool for bioethics and medical ethics and explain how NITs can contribute to policy design in non-ideal circumstances. Different NITs can coexist and be evaluated vis-à-vis the IT. Additionally, I address what an individual doctor ought to do in this non-ideal context with the view that knowledge of NITs can facilitate the decision-making process. NITs help conceptualise problems faced in the context of non-compliance and scarcity in a better and more realistic way. Deciding which policy is optimal in such contexts may influence physicians' decisions regarding their patients. Thus, this analysis-usually identified only with policy making-may also be relevant to medical ethics. Finally, I recognise that this is merely a first step in an unexplored but fundamental theoretical area and that more work needs to be done. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  11. Development of Quenching-qPCR (Q-Q) assay for measuring absolute intracellular cleavage efficiency of ribozyme.

    PubMed

    Kim, Min Woo; Sun, Gwanggyu; Lee, Jung Hyuk; Kim, Byung-Gee

    2018-06-01

    Ribozyme (Rz) is a very attractive RNA molecule in metabolic engineering and synthetic biology fields where RNA processing is required as a control unit or ON/OFF signal for its cleavage reaction. In order to use Rz for such RNA processing, Rz must have highly active and specific catalytic activity. However, current methods for assessing the intracellular activity of Rz have limitations such as difficulty in handling and inaccuracies in the evaluation of correct cleavage activity. In this paper, we proposed a simple method to accurately measure the "intracellular cleavage efficiency" of Rz. This method deactivates unwanted activity of Rz which may consistently occur after cell lysis using DNA quenching method, and calculates the cleavage efficiency by analyzing the cleaved fraction of mRNA by Rz from the total amount of mRNA containing Rz via quantitative real-time PCR (qPCR). The proposed method was applied to measure "intracellular cleavage efficiency" of sTRSV, a representative Rz, and its mutant, and their intracellular cleavage efficiencies were calculated as 89% and 93%, respectively. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H.

    PubMed

    Schultz, Sharon J; Zhang, Miaohua; Champoux, James J

    2010-03-19

    The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3'-end-directed, and RNA 5'-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions -1 and +1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3'-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions +1, -2, -4, -5, -10, and -14 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions -7 and -12 for HIV-1 and positions -4, -9, and -11 for M-MuLV had more modest effects. DNA 3'-end-directed cleavage was influenced substantially by positions +1, -2, -4, and -5 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions +1 and -2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  13. Genetic and environmental influences on thin-ideal internalization across puberty and preadolescent, adolescent, and young adult development.

    PubMed

    Suisman, Jessica L; Thompson, J Kevin; Keel, Pamela K; Burt, S Alexandra; Neale, Michael; Boker, Steven; Sisk, Cheryl; Klump, Kelly L

    2014-11-01

    Mean-levels of thin-ideal internalization increase during adolescence and pubertal development, but it is unknown whether these phenotypic changes correspond to developmental changes in etiological (i.e., genetic and environmental) risk. Given the limited knowledge on risk for thin-ideal internalization, research is needed to guide the identification of specific types of risk factors during critical developmental periods. The present twin study examined genetic and environmental influences on thin-ideal internalization across adolescent and pubertal development. Participants were 1,064 female twins (ages 8-25 years) from the Michigan State University Twin Registry. Thin-ideal internalization and pubertal development were assessed using self-report questionnaires. Twin moderation models were used to examine if age and/or pubertal development moderate genetic and environmental influences on thin-ideal internalization. Phenotypic analyses indicated significant increases in thin-ideal internalization across age and pubertal development. Twin models suggested no significant differences in etiologic effects across development. Nonshared environmental influences were most important in the etiology of thin-ideal internalization, with genetic, shared environmental, and nonshared environmental accounting for approximately 8%, 15%, and 72%, respectively, of the total variance. Despite mean-level increases in thin-ideal internalization across development, the relative influence of genetic versus environmental risk did not differ significantly across age or pubertal groups. The majority of variance in thin-ideal internalization was accounted for by environmental factors, suggesting that mean-level increases in thin-ideal internalization may reflect increases in the magnitude/strength of environmental risk across this period. Replication is needed, particularly with longitudinal designs that assess thin-ideal internalization across key developmental phases. © 2014 Wiley

  14. Senior medical student opinions regarding the ideal urology interview day.

    PubMed

    Jacobs, Jesse C; Guralnick, Michael L; Sandlow, Jay I; Langenstroer, Peter; Begun, Frank P; See, William A; O'Connor, Robert Corey

    2014-01-01

    Applicant interviews for urology residency positions are a stressful and costly process for students, faculty, and staff. We conducted a prospective survey to better determine what urology applicants perceive as an ideal interview process to gain sufficient knowledge about a training program. A questionnaire was anonymously completed by all urology residency applicants interviewing at the Medical College of Wisconsin from 2007 to 2013. Questionnaire subject headings included "ideal interview format," "factors contributing to understanding the residency program," and "factors contributing to final rank list order." Questionnaires were distributed to and completed by 221 senior medical students applying for a urology residency position. Most respondents (>80%) reported they would prefer to partake in 5 to 7 faculty interviews in an office setting with the total interview process spanning half to three-fourths of the workday. Spending time with current residents was considered the most valuable tool to acquire knowledge about a residency program. The most important criteria when ranking a program were resident satisfaction, resident operative experience, and perceived strength of faculty. Academic urology programs may wish to consider applicant ideals when organizing residency interviews. Interaction with current residents appears to be the most valuable resource allowing applicants to garner knowledge about a urology training program. Copyright © 2014 Association of Program Directors in Surgery. Published by Elsevier Inc. All rights reserved.

  15. Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D

    PubMed Central

    Lei, Xiaobo; Zhang, Zhenzhen; Xiao, Xia; Qi, Jianli

    2017-01-01

    ABSTRACT Enterovirus 71 (EV71) can cause hand-foot-and-mouth disease (HFMD) in young children. Severe infection with EV71 can lead to neurological complications and even death. However, the molecular basis of viral pathogenesis remains poorly understood. Here, we report that EV71 induces degradation of gasdermin D (GSDMD), an essential component of pyroptosis. Remarkably, the viral protease 3C directly targets GSDMD and induces its cleavage, which is dependent on the protease activity. Further analyses show that the Q193-G194 pair within GSDMD is the cleavage site of 3C. This cleavage produces a shorter N-terminal fragment spanning amino acids 1 to 193 (GSDMD1–193). However, unlike the N-terminal fragment produced by caspase-1 cleavage, this fragment fails to trigger cell death or inhibit EV71 replication. Importantly, a T239D or F240D substitution abrogates the activity of GSDMD consisting of amino acids 1 to 275 (GSDMD1–275). This is correlated with the lack of pyroptosis or inhibition of viral replication. These results reveal a previously unrecognized strategy for EV71 to evade the antiviral response. IMPORTANCE Recently, it has been reported that GSDMD plays a critical role in regulating lipopolysaccharide and NLRP3-mediated interleukin-1β (IL-1β) secretion. In this process, the N-terminal domain of p30 released from GSDMD acts as an effector in cell pyroptosis. We show that EV71 infection downregulates GSDMD. EV71 3C cleaves GSDMD at the Q193-G194 pair, resulting in a truncated N-terminal fragment disrupted for inducing cell pyroptosis. Notably, GSDMD1–275 (p30) inhibits EV71 replication whereas GSDMD1–193 does not. These results reveal a new strategy for EV71 to evade the antiviral response. PMID:28679757

  16. Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D.

    PubMed

    Lei, Xiaobo; Zhang, Zhenzhen; Xiao, Xia; Qi, Jianli; He, Bin; Wang, Jianwei

    2017-09-15

    Enterovirus 71 (EV71) can cause hand-foot-and-mouth disease (HFMD) in young children. Severe infection with EV71 can lead to neurological complications and even death. However, the molecular basis of viral pathogenesis remains poorly understood. Here, we report that EV71 induces degradation of gasdermin D (GSDMD), an essential component of pyroptosis. Remarkably, the viral protease 3C directly targets GSDMD and induces its cleavage, which is dependent on the protease activity. Further analyses show that the Q193-G194 pair within GSDMD is the cleavage site of 3C. This cleavage produces a shorter N-terminal fragment spanning amino acids 1 to 193 (GSDMD 1-193 ). However, unlike the N-terminal fragment produced by caspase-1 cleavage, this fragment fails to trigger cell death or inhibit EV71 replication. Importantly, a T239D or F240D substitution abrogates the activity of GSDMD consisting of amino acids 1 to 275 (GSDMD 1-275 ). This is correlated with the lack of pyroptosis or inhibition of viral replication. These results reveal a previously unrecognized strategy for EV71 to evade the antiviral response. IMPORTANCE Recently, it has been reported that GSDMD plays a critical role in regulating lipopolysaccharide and NLRP3-mediated interleukin-1β (IL-1β) secretion. In this process, the N-terminal domain of p30 released from GSDMD acts as an effector in cell pyroptosis. We show that EV71 infection downregulates GSDMD. EV71 3C cleaves GSDMD at the Q193-G194 pair, resulting in a truncated N-terminal fragment disrupted for inducing cell pyroptosis. Notably, GSDMD 1-275 (p30) inhibits EV71 replication whereas GSDMD 1-193 does not. These results reveal a new strategy for EV71 to evade the antiviral response. Copyright © 2017 American Society for Microbiology.

  17. Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

    PubMed

    Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

    2012-10-09

    The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

  18. Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein

    PubMed Central

    Welch, Brett D.; Liu, Yuanyuan; Kors, Christopher A.; Leser, George P.; Jardetzky, Theodore S.; Lamb, Robert A.

    2012-01-01

    The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein. PMID:23012473

  19. Hairpin ribozyme cleavage catalyzed by aminoglycoside antibiotics and the polyamine spermine in the absence of metal ions.

    PubMed Central

    Earnshaw, D J; Gait, M J

    1998-01-01

    The hairpin ribozyme is a small catalytic RNA that achieves an active configuration by docking of its two helical domains in an antiparallel fashion. Both docking and subsequent cleavage are dependent on the presence of divalent metal ions, such as magnesium, but there is no evidence to date for direct participation of such ions in the chemical cleavage step. We show that aminoglycoside antibiotics inhibit cleavage of the hairpin ribozyme in the presence of metal ions with the most effective being 5-epi-sisomicin and neomycin B. In contrast, in the absence of metal ions, a number of aminoglycoside antibiotics at 10 mM concentration promote hairpin cleavage with rates only 13-20-fold lower than the magnesium-dependent reaction. We show that neomycin B competes with metal ions by ion replacement with the postively charged amino groups of the antibiotic. In addition, we show that the polyamine spermine at 10 mM promotes efficient hairpin cleavage with rates similar to the magnesium-dependent reaction. Low concentrations of either spermine or the shorter polyamine spermidine synergize with 5 mM magnesium ions to boost cleavage rates considerably. In contrast, at 500 microM magnesium ions, 4 mM spermine, but not spermidine, boosts the cleavage rate. The results have significance both in understanding the role of ions in hairpin ribozyme cleavage and in potential therapeutic applications in mammalian cells. PMID:9837982

  20. Developmental Idealism in China

    PubMed Central

    Thornton, Arland; Xie, Yu

    2016-01-01

    This paper examines the intersection of developmental idealism with China. It discusses how developmental idealism has been widely disseminated within China and has had enormous effects on public policy and programs, on social institutions, and on the lives of individuals and their families. This dissemination of developmental idealism to China began in the 19th century, when China met with several military defeats that led many in the country to question the place of China in the world. By the beginning of the 20th century, substantial numbers of Chinese had reacted to the country’s defeats by exploring developmental idealism as a route to independence, international respect, and prosperity. Then, with important but brief aberrations, the country began to implement many of the elements of developmental idealism, a movement that became especially important following the assumption of power by the Communist Party of China in 1949. This movement has played a substantial role in politics, in the economy, and in family life. The beliefs and values of developmental idealism have also been directly disseminated to the grassroots in China, where substantial majorities of Chinese citizens have assimilated them. These ideas are both known and endorsed by very large numbers in China today. PMID:28316833

  1. Developmental Idealism in China.

    PubMed

    Thornton, Arland; Xie, Yu

    2016-10-01

    This paper examines the intersection of developmental idealism with China. It discusses how developmental idealism has been widely disseminated within China and has had enormous effects on public policy and programs, on social institutions, and on the lives of individuals and their families. This dissemination of developmental idealism to China began in the 19 th century, when China met with several military defeats that led many in the country to question the place of China in the world. By the beginning of the 20 th century, substantial numbers of Chinese had reacted to the country's defeats by exploring developmental idealism as a route to independence, international respect, and prosperity. Then, with important but brief aberrations, the country began to implement many of the elements of developmental idealism, a movement that became especially important following the assumption of power by the Communist Party of China in 1949. This movement has played a substantial role in politics, in the economy, and in family life. The beliefs and values of developmental idealism have also been directly disseminated to the grassroots in China, where substantial majorities of Chinese citizens have assimilated them. These ideas are both known and endorsed by very large numbers in China today.

  2. Wnt5A Activates the Calpain-Mediated Cleavage of Filamin A

    PubMed Central

    O’Connell, Michael P.; Fiori, Jennifer L.; Baugher, Katherine M.; Indig, Fred E.; French, Amanda D.; Camilli, Tura C.; Frank, Brittany P.; Earley, Rachel; Hoek, Keith S.; Hasskamp, Joanne H.; Elias, E. George; Taub, Dennis D.; Bernier, Michel; Weeraratna, Ashani T.

    2009-01-01

    We have previously shown that Wnt5A and ROR2, an orphan tyrosine kinase receptor, interact to mediate melanoma cell motility. In other cell types, this can occur through the interaction of ROR2 with the cytoskeletal protein filamin A. Here, we found that filamin A protein levels correlated with Wnt5A levels in melanoma cells. Small interfering RNA (siRNA) knockdown of WNT5A decreased filamin A expression. Knockdown of filamin A also corresponded to a decrease in melanoma cell motility. In metastatic cells, filamin A expression was predominant in the cytoplasm, which western analysis indicated was due to the cleavage of filamin A in these cells. Treatment of nonmetastatic melanoma cells with recombinant Wnt5A increased filamin A cleavage, and this could be prevented by the knockdown of ROR2 expression. Further, BAPTA-AM chelation of intracellular calcium also inhibited filamin A cleavage, leading to the hypothesis that Wnt5A/ROR2 signaling could cleave filamin A through activation of calcium-activated proteases, such as calpains. Indeed, WNT5A knockdown decreased calpain 1 expression, and by inhibiting calpain 1 either pharmacologically or using siRNA, it decreased cell motility. Our results indicate that Wnt5A activates calpain-1, leading to the cleavage of filamin A, which results in a remodeling of the cytoskeleton and an increase in melanoma cell motility. PMID:19177143

  3. Knowledge-transfer learning for prediction of matrix metalloprotease substrate-cleavage sites.

    PubMed

    Wang, Yanan; Song, Jiangning; Marquez-Lago, Tatiana T; Leier, André; Li, Chen; Lithgow, Trevor; Webb, Geoffrey I; Shen, Hong-Bin

    2017-07-18

    Matrix Metalloproteases (MMPs) are an important family of proteases that play crucial roles in key cellular and disease processes. Therefore, MMPs constitute important targets for drug design, development and delivery. Advanced proteomic technologies have identified type-specific target substrates; however, the complete repertoire of MMP substrates remains uncharacterized. Indeed, computational prediction of substrate-cleavage sites associated with MMPs is a challenging problem. This holds especially true when considering MMPs with few experimentally verified cleavage sites, such as for MMP-2, -3, -7, and -8. To fill this gap, we propose a new knowledge-transfer computational framework which effectively utilizes the hidden shared knowledge from some MMP types to enhance predictions of other, distinct target substrate-cleavage sites. Our computational framework uses support vector machines combined with transfer machine learning and feature selection. To demonstrate the value of the model, we extracted a variety of substrate sequence-derived features and compared the performance of our method using both 5-fold cross-validation and independent tests. The results show that our transfer-learning-based method provides a robust performance, which is at least comparable to traditional feature-selection methods for prediction of MMP-2, -3, -7, -8, -9 and -12 substrate-cleavage sites on independent tests. The results also demonstrate that our proposed computational framework provides a useful alternative for the characterization of sequence-level determinants of MMP-substrate specificity.

  4. Demethylation and cleavage of dimethylsulfoniopropionate in marine intertidal sediments

    USGS Publications Warehouse

    Visscher, P.T.; Kiene, R.P.; Taylor, B.F.

    1994-01-01

    Demethylation and cleavage of dimethylsulfoniopropionate (DMSP) was measured in three different types of,intertidal marine sediments: a cyanobacterial mat, a diatom-covered tidal flat and a carbonate sediment. Consumption rates of added DMSP were highest in cyanobacterial mat slurries (59 ?? mol DMSP l-1 slurry h-1) and lower in slurries from a diatom mat and a carbonate tidal sediment (24 and 9 ??mol DMSP l-1 h-1, respectively). Dimethyl sulfide (DMS) and 3-mercaptopropionate (MPA) were produced simultaneously during DMSP consumption, indicating that cleavage and demethylation occurred at the same time. Viable counts of DMSP-utilizing bacteria revealed a population of 2 x 107 cells cm-3 sediment (90% of these cleaved DMSP to DMS, 10% demethylated DMSP to MPA) in the cyanobacterial mat, 7 x 105 cells cm-3 in the diatom mat (23% cleavers, 77% demethylators), and 9 x 104 cells cm-3 (20% cleavers and 80% demethylators) in the carbonate sediment. In slurries of the diatom mat, the rate of MPA production from added 3-methiolpropionate (MMPA) was 50% of the rate of MPA formation from DMSP. The presence of a large population of demethylating bacteria and the production of MPA from DMSP suggest that the demethylation pathway, in addition to cleavage, contributes significantly to DMSP consumption in coastal sediments.

  5. Kinetic Control in the Cleavage of Unsymmetrical Disilanes.

    PubMed

    Hevesi, Làszlò; Dehon, Michael; Crutzen, Raphael; Lazarescu-Grigore, Adriana

    1997-04-04

    A series of 12 phenyl-substituted arylpentamethyldisilanes 1a-l have been synthesized in order to examine the regioselectivity of their nucleophilic Si,Si bond cleavage reactions under Still's conditions (MeLi/HMPA/0 degrees C). It has been found that the sensitivity of these reactions to the electronic effects of the substituents in the phenyl ring could be described by the Hammett-type equation log(k(A)/k(B)) = 0.4334 + 2.421(Sigmasigma); (correlation coefficient R = 0.983). The k(A)/k(B) ratio represents the relative rate of attack at silicon atom A (linked to the aryl ring) or at silicon atom B (away from the aryl ring) of the unsymmetrical disilanes. Thus, the present investigation shows that the earlier belief according to which the nucleophilic cleavage of unsymmetrical disilanes always produces the more stable silyl anionic species (thermodynamic control) should be abandoned, or at least seriously amended: kinetic factors appear to exert a primary influence on the regioselectivity of such reactions. Since the two major kinetic factors (i.e., electrophilic character of and steric hindrance at a given silicon atom) have opposite effects on the orientation of the reaction, it may happen that kinetic and thermodynamic control lead to the same result. For some of the unsymmetrical disilanes studied, the major reaction path was not the Si,Si bond cleavage; instead, Si-aryl bond breaking occurred, producing the corresponding aryl anions.

  6. A Proposed Method for the Computer-aided Discovery and Design of High-strength, Ductile Metals

    NASA Astrophysics Data System (ADS)

    Winter, Ian Stewart

    Gum Metal, a class of Ti-Nb alloys, has generated a great deal of interest in the metallurgical community since its development in 2003. These alloys display numerous novel and anomalous properties, many of which only occur after severe plastic deformation has been incurred on the material. Such properties include: super-elasticity, super-coldworkability, Invar and Elinvar behavior, high ductility, as well as high strength. The high strength of gum metal has generated particular enthusiasm as it is on the order of the predicted ideal strength of the material. Many of the properties of gum metal appear to be a direct result of tuning the composition to be near an elastic instability resulting in a high degree of elastic anisotropy. This presents an opportunity for the computer-aided discovery and design of structural materials as the ideal strength and elastic anisotropy can be approximated from the elastic constants. Two approaches are described for searching for this high ansitropy. In the first, The possibility of forming gum metal in Mg is explored by tuning the material to be near the BCC-HCP transition either by pressure or alloying with Li. The second makes use of the Materials Project's elastic constants database, which contains thousands of ordered compounds, in order to screen for gum metal candidates. By defining an elastic anisotropy parameter consistent with the behavior of gum metal and calculating it for all cubic materials in the elastic constants database several gum metal candidates are found. In order to better assess their candidacy information on the intrinsic ductility of these materials is necessary. A method is proposed for calculating the ideal strength and deformation mode of a solid solution from first-principles. In order to validate this method the intrinsic ductile-to-brittle transition composition of Ti-V systems is calculated. It is further shown that this method can be applied to the calculation of an ideal tensile yield surface.

  7. Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

    NASA Astrophysics Data System (ADS)

    Jepsen, Morten Leth; Harmsen, Charlotte; Godbole, Adwait Anand; Nagaraja, Valakunja; Knudsen, Birgitta R.; Ho, Yi-Ping

    2015-12-01

    We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes. Electronic supplementary information (ESI) available: Characterization of the QD-based DNA Nanosensor. See DOI: 10.1039/c5nr06326d

  8. Trichomonas vaginalis Metalloproteinase Induces mTOR Cleavage of SiHa Cells

    PubMed Central

    Quan, Juan-Hua; Choi, In-Wook; Yang, Jung-Bo; Zhou, Wei; Cha, Guang-Ho; Zhou, Yu; Ryu, Jae-Sook

    2014-01-01

    Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite. PMID:25548410

  9. Numerical simulations of SHPB experiments for the dynamic compressive strength and failure of ceramics

    NASA Astrophysics Data System (ADS)

    Anderson, Charles E., Jr.; O'Donoghue, Padraic E.; Lankford, James; Walker, James D.

    1992-06-01

    Complementary to a study of the compressive strength of ceramic as a function of strain rate and confinement, numerical simulations of the split-Hopkinson pressure bar (SHPB) experiments have been performed using the two-dimensional wave propagation computer program HEMP. The numerical effort had two main thrusts. Firstly, the interpretation of the experimental data relies on several assumptions. The numerical simulations were used to investigate the validity of these assumptions. The second part of the effort focused on computing the idealized constitutive response of a ceramic within the SHPB experiment. These numerical results were then compared against experimental data. Idealized models examined included a perfectly elastic material, an elastic-perfectly plastic material, and an elastic material with failure. Post-failure material was modeled as having either no strength, or a strength proportional to the mean stress. The effects of confinement were also studied. Conclusions concerning the dynamic behavior of a ceramic up to and after failure are drawn from the numerical study.

  10. Recycling of protein subunits during DNA translocation and cleavage by Type I restriction-modification enzymes

    PubMed Central

    Simons, Michelle; Szczelkun, Mark D.

    2011-01-01

    The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5′-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can ‘turnover’ in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase–nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed. PMID:21712244

  11. Condensed tannins: A novel rearrangement of procyanidins and prodelphinidins in thiolytic cleavage

    Treesearch

    G. Wayne McGraw; Jan P. Steynberg; Richard W. Hemingway

    1993-01-01

    Conditions commonly used for the thiolytic cleavage of interflavanoid bonds of condensed tannins also result in cleavage of the C4 to C10 bond of flavan units. Subsequenet lectrophilic attack of the C4 carbocation on the C2' or C6' of the B-ring, and loss of phloroglucinol (the A-ring), result in the formation of a mixture of 1,3-dithiobenzyl-2,4,s,6-...

  12. Inflammatory Caspases: Activation and Cleavage of Gasdermin-D In Vitro and During Pyroptosis.

    PubMed

    Zhao, Yue; Shi, Jianjin; Shao, Feng

    2018-01-01

    Gasdermin-D (also known as GSDMD), the newly identified executioner of pyroptotic cell death, is cleaved by activated caspase-1 downstream of canonical inflammasome activation or caspase-4, 5, and 11 upon their ligation and activation by cytosolic LPS. Upon a single cleavage between the two domains in Gasdermin-D, the N-terminal domain binds to membrane lipids and lyses cells by forming pores of an inner diameter of 10-14 nm within the membrane. The inter-domain cleavage of Gasdermin-D is a reliable marker for the activation of inflammatory caspases and cell pyroptosis. Here, we describe the methods for examining Gasdermin-D cleavage by activated inflammatory caspases in vitro and upon inflammasome activation in vivo.

  13. Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate.

    PubMed

    Chang, J Y

    1985-09-02

    alpha-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative amino-terminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of alpha-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by alpha-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit alpha-thrombin in 20 microliters of 50 mM ammonium bicarbonate at 25 degrees C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for alpha-thrombin has the structures of (a) P4-P3-Pro-Arg-P1'-P2', where P3 and P4 are hydrophobic amino acid and P1', P2' are nonacidic amino acids and (b) P2-Arg-P1', where P2 or P1' are Gly. The requirement for hydrophobic P3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by alpha-thrombin can be reasonably predicted and that chemical modification can be a useful tool in enhancing

  14. Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage.

    PubMed

    Lavoie, Mathieu; Abou Elela, Sherif

    2008-08-19

    Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.

  15. Genetic and Environmental Influences on Thin-Ideal Internalization across Puberty and Pre-Adolescent, Adolescent, and Young Adult Development

    PubMed Central

    Suisman, Jessica L.; Thompson, J. Kevin; Keel, Pamela K.; Burt, S. Alexandra; Neale, Michael; Boker, Steven; Sisk, Cheryl; Klump, Kelly L.

    2014-01-01

    Objective Mean-levels of thin-ideal internalization increase during adolescence and pubertal development, but it is unknown whether these phenotypic changes correspond to developmental changes in etiological (i.e., genetic and environmental) risk. Given the limited knowledge on risk for thin-ideal internalization, research is needed to guide the identification of specific types of risk factors during critical developmental periods. The present twin study examined genetic and environmental influences on thin-ideal internalization across adolescent and pubertal development. Method Participants were 1,064 female twins (ages 8–25 years) from the Michigan State University Twin Registry. Thin-ideal internalization and pubertal development were assessed using self-report questionnaires. Twin moderation models were used to examine if age and/or pubertal development moderate genetic and environmental influences on thin-ideal internalization. Results Phenotypic analyses indicated significant increases in thin-ideal internalization across age and pubertal development. Twin models suggested no significant differences in etiologic effects across development. Nonshared environmental influences were most important in the etiology of thin-ideal internalization, with genetic, shared environmental, and nonshared environmental accounting for approximately 8%, 15%, and 72%, respectively, of the total variance. Discussion Despite mean-level increases in thin-ideal internalization across development, the relative influence of genetic versus environmental risk did not differ significantly across age or pubertal groups. The majority of variance in thin-ideal internalization was accounted for by environmental factors, suggesting that mean-level increases in thin-ideal internalization may reflect increases in the magnitude/strength of environmental risk across this period. Replication is needed, particularly with longitudinal designs that assess thin-ideal internalization across key

  16. Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system.

    PubMed

    Liu, Xiaoxi; Homma, Ayaka; Sayadi, Jamasb; Yang, Shu; Ohashi, Jun; Takumi, Toru

    2016-01-27

    The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.

  17. Advances in cleavage fracture modelling in steels: Micromechanical, numerical and multiscale aspects

    NASA Astrophysics Data System (ADS)

    Pineau, André; Tanguy, Benoît

    2010-04-01

    Brittle cleavage fracture remains one of the major concerns for structural integrity assessment. The main characteristics of this mode of failure in relation to the stress field ahead of a crack, tip are described in the introduction. The emphasis is laid on the physical origins of scatter and the size effect observed in ferritic steels. It is shown that cleavage fracture is controlled by physical events occurring at different scales: initiation at (sub)micrometric particles, propagation across grain boundaries (10-50 microns) and final fracture at centimetric scale. The two first scales are detailed in this paper. The statistical origin of cleavage is described quantitatively from both microstructural defects and stress-strain heterogeneities due to crystalline plasticity at the grain scale. Existing models are applied to the prediction of the variation of Charpy fracture toughness with temperature.

  18. Sequence-dependent DNA flexibility mediates DNase I cleavage.

    PubMed

    Heddi, Brahim; Abi-Ghanem, Josephine; Lavigne, Marc; Hartmann, Brigitte

    2010-01-08

    Understanding the preference of nonspecific proteins for certain DNA structural features requires an accurate description of the properties of free DNA, especially regarding their possible predisposition to adopt a conformation that favors the formation of a complex. Exploiting previous exhaustive NMR studies performed on free DNA oligomers, we investigated the molecular basis of DNase I sensitivity under conditions where DNase I binding limits the probability of cleavage. We showed that cleavage intensity was correlated with adjacent 3' phosphate linkage flexibility, monitored by (31)P chemical shifts. Examining NMR-refined DNA structures highlighted that sequence-dependent flexible phosphates were associated with large minor groove variations that may promote the affinity of DNase I, according to relevant DNA-protein complexes. In sum, this work demonstrates that specificity in DNA-DNase I interaction is mediated by DNA flexibility, which influences the induced-fit transitions required to form productive complexes.

  19. Embryonic Cleavage Cycles: How Is a Mouse Like a Fly?

    PubMed Central

    O’Farrell, Patrick H.; Stumpff, Jason; Su, Tin Tin

    2009-01-01

    The evolutionary advent of uterine support of embryonic growth in mammals is relatively recent. Nonetheless, striking differences in the earliest steps of embryogenesis make it difficult to draw parallels even with other chordates. We suggest that use of fertilization as a reference point misaligns the earliest stages and masks parallels that are evident when development is aligned at conserved stages surrounding gastrulation. In externally deposited eggs from representatives of all the major phyla, gastrulation is preceded by specialized extremely rapid cleavage cell cycles. Mammals also exhibit remarkably fast cell cycles in close association with gastrulation, but instead of beginning development with these rapid cycles, the mammalian egg first devotes itself to the production of extraembryonic structures. Previous attempts to identify common features of cleavage cycles focused on post-fertilization divisions of the mammalian egg. We propose that comparison to the rapid peri-gastrulation cycles is more appropriate and suggest that these cycles are related by evolutionary descent to the early cleavage stages of embryos such as those of frog and fly. The deferral of events in mammalian embryogenesis might be due to an evolutionary shift in the timing of fertilization. PMID:14711435

  20. Polycystin-1 C-terminal Cleavage Is Modulated by Polycystin-2 Expression*

    PubMed Central

    Bertuccio, Claudia A.; Chapin, Hannah C.; Cai, Yiqiang; Mistry, Kavita; Chauvet, Veronique; Somlo, Stefan; Caplan, Michael J.

    2009-01-01

    Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway. PMID:19491093

  1. Cnidarian microRNAs frequently regulate targets by cleavage.

    PubMed

    Moran, Yehu; Fredman, David; Praher, Daniela; Li, Xin Z; Wee, Liang Meng; Rentzsch, Fabian; Zamore, Phillip D; Technau, Ulrich; Seitz, Hervé

    2014-04-01

    In bilaterians, which comprise most of extant animals, microRNAs (miRNAs) regulate the majority of messenger RNAs (mRNAs) via base-pairing of a short sequence (the miRNA "seed") to the target, subsequently promoting translational inhibition and transcript instability. In plants, many miRNAs guide endonucleolytic cleavage of highly complementary targets. Because little is known about miRNA function in nonbilaterian animals, we investigated the repertoire and biological activity of miRNAs in the sea anemone Nematostella vectensis, a representative of Cnidaria, the sister phylum of Bilateria. Our work uncovers scores of novel miRNAs in Nematostella, increasing the total miRNA gene count to 87. Yet only a handful are conserved in corals and hydras, suggesting that microRNA gene turnover in Cnidaria greatly exceeds that of other metazoan groups. We further show that Nematostella miRNAs frequently direct the cleavage of their mRNA targets via nearly perfect complementarity. This mode of action resembles that of small interfering RNAs (siRNAs) and plant miRNAs. It appears to be common in Cnidaria, as several of the miRNA target sites are conserved among distantly related anemone species, and we also detected miRNA-directed cleavage in Hydra. Unlike in bilaterians, Nematostella miRNAs are commonly coexpressed with their target transcripts. In light of these findings, we propose that post-transcriptional regulation by miRNAs functions differently in Cnidaria and Bilateria. The similar, siRNA-like mode of action of miRNAs in Cnidaria and plants suggests that this may be an ancestral state.

  2. Non-T cell activation linker (NTAL) proteolytic cleavage as a terminator of activatory intracellular signals.

    PubMed

    Arbulo-Echevarria, Mikel M; Muñoz-Miranda, Juan Pedro; Caballero-García, Andrés; Poveda-Díaz, José L; Fernández-Ponce, Cecilia; Durán-Ruiz, M Carmen; Miazek, Arkadiusz; García-Cózar, Francisco; Aguado, Enrique

    2016-08-01

    Non-T cell activation linker is an adaptor protein that is tyrosine phosphorylated upon cross-linking of immune receptors expressed on B lymphocytes, NK cells, macrophages, basophils, or mast cells, allowing the recruitment of cytosolic mediators for downstream signaling pathways. Fas receptor acts mainly as a death receptor, and when cross-linked with Fas ligand, many proteins are proteolytically cleaved, including several signaling molecules in T and B cells. Fas receptor triggering also interferes with TCR intracellular signals, probably by means of proteolytic cleavage of several adaptor proteins. We have previously found that the adaptor linker for activation of T cells, evolutionarily related to non-T cell activation linker, is cleaved upon proapoptotic stimuli in T lymphocytes and thymocytes, in a tyrosine phosphorylation-dependent fashion. Here, we describe non-T cell activation linker proteolytic cleavage triggered in human B cells and monocytes by Fas cross-linking and staurosporine treatment. Non-T cell activation linker is cleaved, producing an N-terminal fragment of ∼22 kDa, and such cleavage is abrogated in the presence of caspase 8/granzyme B and caspase 3 inhibitors. Moreover, we have identified an aspartic acid residue at which non-T cell activation linker is cleaved, which similar to linker for activation of T cells, this aspartic acid residue is located close to tyrosine and serine residues, suggesting an interdependence of phosphorylation and proteolytic cleavage. Consistently, induction of non-T cell activation linker phosphorylation by pervanadate inhibits its cleavage. Interestingly, the truncated isoform of non-T cell activation linker, generated after cleavage, has a decreased signaling ability when compared with the full-length molecule. Altogether, our results suggest that cleavage of transmembrane adaptors constitutes a general mechanism for signal termination of immune receptors. © Society for Leukocyte Biology.

  3. Application of Strength Requirements to Complex Loading Scenarios

    NASA Technical Reports Server (NTRS)

    England, Scott; Rajulu, Sudhakar

    2016-01-01

    NASA's endeavors in human spaceflight rely on extensive volumes of human-systems integration requirements to ensure mission success. These requirements protect for space hardware accommodation for the full range of potential crewmembers, but cannot cover every possible action and contingency in detail. This study was undertaken in response to questions from various strength requirement users who were unclear how to apply idealized strength requirements that did not map well to the complex loading scenarios that crewmembers would encounter. Three of the most commonly occurring questions from stakeholders were selected to be investigated with human testing and human modeling. Preliminary findings indicate deviation from nominal postures can affect strength requirement compliance positively or negatively, depending on the nature of the deviation. Human modeling offers some avenues for quickly addressing requirement verification questions, but is limited by the fidelity of the model and environment.

  4. Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

    PubMed

    Kukiełka, E; Cederbaum, A I

    1995-04-15

    Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

  5. RNase L Cleavage Products Promote Switch from Autophagy to Apoptosis by Caspase-Mediated Cleavage of Beclin-1

    PubMed Central

    Siddiqui, Mohammad Adnan; Mukherjee, Sushovita; Manivannan, Praveen; Malathi, Krishnamurthy

    2015-01-01

    Autophagy and apoptosis share regulatory molecules enabling crosstalk in pathways that affect cellular homeostasis including response to viral infections and survival of tumor cells. Ribonuclease L (RNase L) is an antiviral endonuclease that is activated in virus-infected cells and cleaves viral and cellular single-stranded RNAs to produce small double-stranded RNAs with roles in amplifying host responses. Activation of RNase L induces autophagy and apoptosis in many cell types. However, the mechanism by which RNase L mediates crosstalk between these two pathways remains unclear. Here we show that small dsRNAs produced by RNase L promote a switch from autophagy to apoptosis by caspase-mediated cleavage of Beclin-1, terminating autophagy. The caspase 3-cleaved C-terminal fragment of Beclin-1 enhances apoptosis by translocating to the mitochondria along with proapoptotic protein, Bax, and inducing release of cytochrome C to the cytosol. Cleavage of Beclin-1 determines switch to apoptosis since expression of caspase-resistant Beclin-1 inhibits apoptosis and sustains autophagy. Moreover, inhibiting RNase L-induced autophagy promotes cell death and inhibiting apoptosis prolongs autophagy in a cross-inhibitory mechanism. Our results demonstrate a novel role of RNase L generated small RNAs in cross-talk between autophagy and apoptosis that impacts the fate of cells during viral infections and cancer. PMID:26263979

  6. Cleavage reaction of HDV ribozymes in the presence of Mg2+ is accompanied by a conformational change.

    PubMed

    Tanaka, Yoichiro; Tagaya, Mitsuhiro; Hori, Tamaki; Sakamoto, Taiichi; Kurihara, Yasuyuki; Katahira, Masato; Uesugi, Seiichi

    2002-06-01

    Hepatitis delta virus (HDV) ribozymes cleave RNA in the presence of divalent metal ions. We have previously elucidated the solution conformation of a minimized trans-acting HDV ribozyme and obtained evidence by NMR study that an Mg2+ ion binds to a site close to the cleavage site. We examined two ribozyme systems: a pre-cleavage complex with a non-cleavable substrate analogue (mS8) and a post-cleavage complex with a 3' cleavage product (P7). Upon titration with MgCl2, the complex with P7 showed a profound spectral change, while that with mS8 showed broadening of the signals. Analysis of the NOESY spectra of the P7 complex at high Mg2+ concentration revealed that a G:U pair is formed within the L3 loop, and the P1 and P4 stems are stabilized with respect to those of the pre-cleavage complex. The present analysis indicates that the cleavage reaction of the HDV ribozyme produces a big conformational change. Furthermore, presence of the 5'-terminal cytidine residue prevents this conformational change and its absence stabilizes the product-ribozyme complex in the presence of Mg2+. The structure of the Mg2+-bound P7 complex is similar to the crystal structure found for a product-ribozyme complex but is different from the pre-cleavage structure.

  7. Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

    PubMed Central

    Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

    2008-01-01

    The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

  8. Cleavage and polyadenylation: Ending the message expands gene regulation

    PubMed Central

    Neve, Jonathan

    2017-01-01

    ABSTRACT Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3′end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease. PMID:28453393

  9. Longitudinal cleavage of the penis in chronic spinal cord injury: two case reports

    PubMed Central

    Mansoor, Sahibzada Nasir; Ayaz, Saeed Bin; New, Peter

    2016-01-01

    Context Penile cleavage is a rare complication of spinal cord injury (SCI) in patients with a chronic indwelling catheter. We report two cases of chronic SCI who developed penile urethral cleavage after prolonged use of an indwelling catheter for bladder management. Findings A 25-year-old wheelchair mobile male with T7 American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade A paraplegia developed a 4 × 1.5 cm ventral urethral cleavage after using an indwelling catheter for four months with inadequate care. He had an associated urinary tract infection and undiagnosed diabetes mellitus. A suprapubic catheter was inserted and surgical repair recommended after resolution of UTI and adequate control of his diabetes mellitus. After initial treatment he was lost to follow-up. The second patient was a 15-year-old male with AIS grade B tetraplegia who presented with a 2.5 cm cleavage on the ventral aspect of penis for the preceding three months. He had been using an indwelling catheter for bladder management for the previous 18 months. He had modified Ashworth scale grade III spasticity in lower limbs resistant to conservative management. There was no history of trauma, infection or diabetes mellitus. The patient was advised penile urethral repair surgery but was lost to follow-up. Conclusion Penile cleavage is a rare complication of neurogenic bladder in SCI patients. Patients and care givers should be trained in proper bladder management techniques during the hospital stay, counseled regarding the need for regular follow up, and be taught identification and prevention of common complications. PMID:26108452

  10. Longitudinal cleavage of the penis in chronic spinal cord injury: two case reports.

    PubMed

    Mansoor, Sahibzada Nasir; Ayaz, Saeed Bin; Rathore, Farooq Azam; New, Peter

    2016-05-01

    Penile cleavage is a rare complication of spinal cord injury (SCI) in patients with a chronic indwelling catheter. We report two cases of chronic SCI who developed penile urethral cleavage after prolonged use of an indwelling catheter for bladder management. A 25-year-old wheelchair mobile male with T7 American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade A paraplegia developed a 4 × 1.5 cm ventral urethral cleavage after using an indwelling catheter for four months with inadequate care. He had an associated urinary tract infection and undiagnosed diabetes mellitus. A suprapubic catheter was inserted and surgical repair recommended after resolution of UTI and adequate control of his diabetes mellitus. After initial treatment he was lost to follow-up. The second patient was a 15-year-old male with AIS grade B tetraplegia who presented with a 2.5 cm cleavage on the ventral aspect of penis for the preceding three months. He had been using an indwelling catheter for bladder management for the previous 18 months. He had modified Ashworth scale grade III spasticity in lower limbs resistant to conservative management. There was no history of trauma, infection or diabetes mellitus. The patient was advised penile urethral repair surgery but was lost to follow-up. Penile cleavage is a rare complication of neurogenic bladder in SCI patients. Patients and care givers should be trained in proper bladder management techniques during the hospital stay, counseled regarding the need for regular follow up, and be taught identification and prevention of common complications.

  11. Ultimate Longitudinal Strength of Composite Ship Hulls

    NASA Astrophysics Data System (ADS)

    Zhang, Xiangming; Huang, Lingkai; Zhu, Libao; Tang, Yuhang; Wang, Anwen

    2017-01-01

    A simple analytical model to estimate the longitudinal strength of ship hulls in composite materials under buckling, material failure and ultimate collapse is presented in this paper. Ship hulls are regarded as assemblies of stiffened panels which idealized as group of plate-stiffener combinations. Ultimate strain of the plate-stiffener combination is predicted under buckling or material failure with composite beam-column theory. The effects of initial imperfection of ship hull and eccentricity of load are included. Corresponding longitudinal strengths of ship hull are derived in a straightforward method. A longitudinally framed ship hull made of symmetrically stacked unidirectional plies under sagging is analyzed. The results indicate that present analytical results have a good agreement with FEM method. The initial deflection of ship hull and eccentricity of load can dramatically reduce the bending capacity of ship hull. The proposed formulations provide a simple but useful tool for the longitudinal strength estimation in practical design.

  12. Kinetics of acid-catalyzed cleavage of procyanindins

    Treesearch

    Richard W. Hemingway; Gerald W. McGraw

    1983-01-01

    Comparison of the rates of cleavage of isomeric procyanidin dimers in the presence of excess phenylmethane thiol and acetic acid showed that compounds with a C(4)-C(8) interflavanoid bond were cleaved more rapidly than their C(4)-C(6) linked isomers, that 2,3-cis isomers with an axial flavan substituent were cleaved more-rapidly than a 2,3-...

  13. Signal peptide discrimination and cleavage site identification using SVM and NN.

    PubMed

    Kazemian, H B; Yusuf, S A; White, K

    2014-02-01

    About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification. The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification. The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model. © 2013 Published by Elsevier Ltd.

  14. Ideal heat transfer conditions for tubular solar receivers with different design constraints

    NASA Astrophysics Data System (ADS)

    Kim, Jin-Soo; Potter, Daniel; Gardner, Wilson; Too, Yen Chean Soo; Padilla, Ricardo Vasquez

    2017-06-01

    The optimum heat transfer condition for a tubular type solar receiver was investigated for various receiver pipe size, heat transfer fluid, and design requirement and constraint(s). Heat transfer of a single plain receiver pipe exposed to concentrated solar energy was modelled along the flow path of the heat transfer fluid. Three different working fluids, molten salt, sodium, and supercritical carbon dioxide (sCO2) were considered in the case studies with different design conditions. The optimized ideal heat transfer condition was identified through fast iterative heat transfer calculations solving for all relevant radiation, conduction and convection heat transfers throughout the entire discretized tubular receiver. The ideal condition giving the best performance was obtained by finding the highest acceptable solar energy flux optimally distributed to meet different constraint(s), such as maximum allowable material temperature of receiver, maximum allowable film temperature of heat transfer fluid, and maximum allowable stress of receiver pipe material. The level of fluid side turbulence (represented by pressure drop in this study) was also optimized to give the highest net power production. As the outcome of the study gives information on the most ideal heat transfer condition, it can be used as a useful guideline for optimal design of a real receiver and solar field in a combined manner. The ideal heat transfer condition is especially important for high temperature tubular receivers (e.g. for supplying heat to high efficiency Brayton cycle turbines) where the system design and performance is tightly constrained by the receiver pipe material strength.

  15. Cleavage and shedding of E-cadherin after induction of apoptosis.

    PubMed

    Steinhusen, U; Weiske, J; Badock, V; Tauber, R; Bommert, K; Huber, O

    2001-02-16

    Apoptotic cell death induces dramatic molecular changes in cells, becoming apparent on the structural level as membrane blebbing, condensation of the cytoplasm and nucleus, and loss of cell-cell contacts. The activation of caspases is one of the fundamental steps during programmed cell death. Here we report a detailed analysis of the fate of the Ca(2+)-dependent cell adhesion molecule E-cadherin in apoptotic epithelial cells and show that during apoptosis fragments of E-cadherin with apparent molecular masses of 24, 29, and 84 kDa are generated by two distinct proteolytic activities. In addition to a caspase-3-mediated cleavage releasing the cytoplasmic domain of E-cadherin, a metalloproteinase sheds the extracellular domain from the cell surface during apoptosis. Immunofluorescence analysis confirmed that concomitant with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulates in the cytosol. In the presence of inhibitors of caspase-3 and/or metalloproteinases, cleavage of E-cadherin was almost completely blocked. The simultaneous cleavage of the intracellular and extracellular domains of E-cadherin may provide a highly efficient mechanism to disrupt cadherin-mediated cell-cell contacts in apoptotic cells, a prerequisite for cell rounding and exit from the epithelium.

  16. Scales of equilibrium and disequilibrium during cleavage formation in chlorite and biotite-grade phyllites, SE Vermont

    USGS Publications Warehouse

    McWilliams, C.K.; Wintsch, R.P.; Kunk, Michael J.

    2007-01-01

    Detailed electron microprobe analyses of phyllosilicates in crenulated phyllites from south-eastern Vermont show that grain-scale zoning is common, and sympathetic zoning in adjacent minerals is nearly universal. We interpret this to reflect a pressure-solution mechanism for cleavage development, where precipitation from a very small fluid reservoir fractionated that fluid. Multiple analyses along single muscovite, biotite and chlorite grains (30–200 μm in length) show zoning patterns indicating Tschermakitic substitutions in muscovite and both Tschermakitic and di/trioctahedral substitutions in biotite and chlorite. Using cross-cutting relationships and mineral chemistry it is shown that these patterns persist in cleavages produced at metamorphic conditions of chlorite-grade, chlorite-grade overprinted by biotite-grade and biotite-grade. Zoning patterns are comparable in all three settings, requiring a similar cleavage-forming mechanism independent of metamorphic grade. Moreover, the use of 40Ar/39Ar geochronology demonstrates this is true regardless of age. Furthermore, samples with chlorite-grade cleavages overprinted by biotite porphyroblasts suggest the closure temperatures for the diffusion of Al, Si, Mg and Fe ions are greater than the temperature of the biotite isograd (>∼400 °C). Parallel and smoothly fanning tie lines produced by coexisting muscovite–chlorite, and muscovite–biotite pairs on compositional diagrams demonstrate effectively instantaneous chemical equilibrium and probably indicate simultaneous crystallization.These results do not support theories suggesting cleavages form in fluid-dominated systems. If crenulation cleavages formed in systems in which the chemical potentials of all major components are fixed by an external reservoir, then the compositions of individual grains defining these cleavages would be uniform. On the contrary, the fine-scale chemical zoning observed probably reflects a grain-scale process consistent with a

  17. Effect of Loading Rates and Surface Conditions on the Flexural Strength of Borosilicate Glass

    DTIC Science & Technology

    2009-01-01

    strength of etched soda - lime glass rods.32 According to this model, an idealized surface crack is uniformly attacked by acid at every point so that this...R. Lin, ‘‘Effect of Polymer Coatings on the Strength and Fatigue Behavior of Indented Soda - Lime Glass ,’’ Glass Technol., 32 [2] 51–4 (1991). 10J. J...Scott Glaesemann, K. Jakus, and J. E. Ritter Jr., ‘‘Strength Variability of Indented Soda - Lime Glass ,’’ J. Am. Ceram. Soc., 70 [6] 441–4 (1987). 12C

  18. Resistance of Actin to Cleavage during Apoptosis

    NASA Astrophysics Data System (ADS)

    Song, Qizhong; Wei, Tie; Lees-Miller, Susan; Alnemri, Emad; Watters, Dianne; Lavin, Martin F.

    1997-01-01

    A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1β -converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.

  19. Drive for muscularity and social physique anxiety mediate the perceived ideal physique muscle dysmorphia relationship.

    PubMed

    Thomas, Adam; Tod, David A; Edwards, Christian J; McGuigan, Michael R

    2014-12-01

    This study examined the mediating role of drive for muscularity and social physique anxiety (SPA) in the perceived muscular male ideal physique and muscle dysmorphia relationship in weight training men. Men (N = 146, mean ± SD; age, 22.8 ± 5.0 years; weight, 82.0 ± 11.1 kg; height, 1.80 ± 0.07 m; body mass index, 25.1 ± 3.0) who participated in weight training completed validated questionnaires measuring drive for muscularity, SPA, perceived muscular male ideal physique, global muscle dysmorphia, and several characteristics of muscle dysmorphia (exercise dependence, diet manipulation, concerns about size/symmetry, physique protection behavior, and supplementation). Perceived ideal physique was an independent predictor of muscle dysmorphia measures except physique protection (coefficients = 0.113-0.149, p ≤ 0.05). Perceived ideal physique also predicted muscle dysmorphia characteristics (except physique protection and diet) through the indirect drive for muscularity pathway (coefficients = 0.055-0.116, p ≤ 0.05). Perceived ideal physique also predicted size/symmetry concerns and physique protection through the indirect drive for muscularity and SPA pathway (coefficients = 0.080-0.025, p ≤ 0.05). These results extend current research by providing insights into the way correlates of muscle dysmorphia interact to predict the condition. The results also highlight signs (e.g., anxiety about muscularity) that strength and conditioning coaches can use to identify at-risk people who may benefit from being referred for psychological assistance.

  20. Programmable RNA recognition and cleavage by CRISPR/Cas9.

    PubMed

    O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

    2014-12-11

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

  1. Programmable RNA recognition and cleavage by CRISPR/Cas9

    PubMed Central

    O’Connell, Mitchell R.; Oakes, Benjamin L.; Sternberg, Samuel H.; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A.

    2014-01-01

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage1-5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA4-7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition. PMID:25274302

  2. From the ideal market to the ideal clinic: constructing a normative standard of fairness for human subjects research.

    PubMed

    Phillips, Trisha

    2011-02-01

    Preventing exploitation in human subjects research requires a benchmark of fairness against which to judge the distribution of the benefits and burdens of a trial. This paper proposes the ideal market and its fair market price as a criterion of fairness. The ideal market approach is not new to discussions about exploitation, so this paper reviews Wertheimer's inchoate presentation of the ideal market as a principle of fairness, attempt of Emanuel and colleagues to apply the ideal market to human subjects research, and Ballantyne's criticisms of both the ideal market and the resulting benchmark of fairness. It argues that the criticism of this particular benchmark is on point, but the rejection of the ideal market is mistaken. After presenting a complete account of the ideal market, this paper proposes a new method for applying the ideal market to human subjects research and illustrates the proposal by considering a sample case.

  3. A field like today's? The strength of the geomagnetic field 1.1 billion years ago

    NASA Astrophysics Data System (ADS)

    Sprain, Courtney J.; Swanson-Hysell, Nicholas L.; Fairchild, Luke M.; Gaastra, Kevin

    2018-06-01

    Palaeomagnetic data from ancient rocks are one of the few types of observational data that can be brought to bear on the long-term evolution of Earth's core. A recent compilation of palaeointensity estimates from throughout Earth history has been interpreted to indicate that Earth's magnetic field strength increased in the Mesoproterozoic (between 1.5 and 1.0 billion years ago), with this increase taken to mark the onset of inner core nucleation. However, much of the data within the Precambrian palaeointensity database are from Thellier-style experiments with non-ideal behaviour that manifests in results such as double-slope Arai plots. Choices made when interpreting these data may significantly change conclusions about long-term trends in the intensity of Earth's geomagnetic field. In this study, we present new palaeointensity results from volcanics of the ˜1.1-billion-year-old North American Midcontinent Rift. While most of the results exhibit non-ideal double-slope or sagging behaviour in Arai plots, some flows have more ideal single-slope behaviour leading to palaeointensity estimates that may be some of the best constraints on the strength of Earth's field for this time. Taken together, new and previously published palaeointensity data from the Midcontinent Rift yield a median field strength estimate of 56.0 ZAm2—very similar to the median for the past 300 Myr. These field strength estimates are distinctly higher than those for the preceding billion years (Ga) after excluding ca. 1.3 Ga data that may be biased by non-ideal behaviour—consistent with an increase in field strength in the late Mesoproterozoic. However, given that ˜90 per cent of palaeointensity estimates from 1.1 to 0.5 Ga come from the Midcontinent Rift, it is difficult to evaluate whether these high values relative to those estimated for the preceding billion years are the result of a stepwise, sustained increase in dipole moment. Regardless, palaeointensity estimates from the Midcontinent

  4. A field like today's? The strength of the geomagnetic field 1.1 billion years ago

    NASA Astrophysics Data System (ADS)

    Sprain, Courtney J.; Swanson-Hysell, Nicholas L.; Fairchild, Luke M.; Gaastra, Kevin

    2018-02-01

    Paleomagnetic data from ancient rocks are one of the few types of observational data that can be brought to be bear on the long-term evolution of Earth's core. A recent compilation of paleointensity estimates from throughout Earth history has been interpreted to indicate that Earth's magnetic field strength increased in the Mesoproterozoic (between 1.5 and 1.0 billion years ago), with this increase taken to mark the onset of inner core nucleation. However, much of the data within the Precambrian paleointensity database are from Thellier-style experiments with non-ideal behavior that manifests in results such as double-slope Arai plots. Choices made when interpreting these data may significantly change conclusions about long-term trends in the intensity of Earth's geomagnetic field. In this study, we present new paleointensity results from volcanics of the ˜1.1 billion-year-old North American Midcontinent Rift. While most of the results exhibit non-ideal double-slope or sagging behavior in Arai plots, some flows have more ideal single-slope behavior leading to paleointensity estimates that may be some of the best constraints on the strength of Earth's field for this time. Taken together, new and previously published paleointensity data from the Midcontinent Rift yield a median field strength estimate of 56.0 ZAm2—very similar to the median for the past 300 million years. These field strength estimates are distinctly higher than those for the preceding billion years after excluding ca. 1.3 Ga data that may be biased by non-ideal behavior—consistent with an increase in field strength in the late Mesoproterozoic. However, given that ˜90 per cent of paleointensity estimates from 1.1 to 0.5 Ga come from the Midcontinent Rift, it is difficult to evaluate whether these high values relative to those estimated for the preceding billion years are the result of a stepwise, sustained increase in dipole moment. Regardless, paleointensity estimates from the Midcontinent

  5. The action of the bacterial toxin microcin B17. Insight into the cleavage-religation reaction of DNA gyrase.

    PubMed

    Pierrat, Olivier A; Maxwell, Anthony

    2003-09-12

    We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.

  6. Polycystin-1 Cleavage and the Regulation of Transcriptional Pathways

    PubMed Central

    Merrick, David; Bertuccio, Claudia A.; Chapin, Hannah C.; Lal, Mark; Chauvet, Veronique; Caplan, Michael J.

    2013-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end stage renal disease, affecting ~1 in 1,000 people. The disease is characterized by the development of numerous large fluid filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, polycystin-1 and polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments that manifest biological activities. Recent results suggest that the production of polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein. PMID:23824180

  7. Global identification of target recognition and cleavage by the Microprocessor in human ES cells

    PubMed Central

    Seong, Youngmo; Lim, Do-Hwan; Kim, Augustine; Seo, Jae Hong; Lee, Young Sik; Song, Hoseok; Kwon, Young-Soo

    2014-01-01

    The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein–RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3′ overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells. PMID:25326327

  8. Global identification of target recognition and cleavage by the Microprocessor in human ES cells.

    PubMed

    Seong, Youngmo; Lim, Do-Hwan; Kim, Augustine; Seo, Jae Hong; Lee, Young Sik; Song, Hoseok; Kwon, Young-Soo

    2014-11-10

    The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3' overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Ideals as Anchors for Relationship Experiences

    PubMed Central

    Frye, Margaret; Trinitapoli, Jenny

    2016-01-01

    Research on young-adult sexuality in sub-Saharan Africa typically conceptualizes sex as an individual-level risk behavior. We introduce a new approach that connects the conditions surrounding the initiation of sex with subsequent relationship well-being, examines relationships as sequences of interdependent events, and indexes relationship experiences to individually held ideals. New card-sort data from southern Malawi capture young women’s relationship experiences and their ideals in a sequential framework. Using optimal matching, we measure the distance between ideal and experienced relationship sequences to (1) assess the associations between ideological congruence and perceived relationship well-being, (2) compare this ideal-based approach to other experience-based alternatives, and (3) identify individual- and couple-level correlates of congruence between ideals and experiences in the romantic realm. We show that congruence between ideals and experiences conveys relationship well-being along four dimensions: expressions of love and support, robust communication habits, perceived biological safety, and perceived relationship stability. We further show that congruence is patterned by socioeconomic status and supported by shared ideals within romantic dyads. We argue that conceiving of ideals as anchors for how sexual experiences are manifest advances current understandings of romantic relationships, and we suggest that this approach has applications for other domains of life. PMID:27110031

  10. Ideals and Category Typicality

    ERIC Educational Resources Information Center

    Kim, ShinWoo; Murphy, Gregory L.

    2011-01-01

    Barsalou (1985) argued that exemplars that serve category goals become more typical category members. Although this claim has received support, we investigated (a) whether categories have a single ideal, as negatively valenced categories (e.g., cigarette) often have conflicting goals, and (b) whether ideal items are in fact typical, as they often…

  11. Ideal AFROC and FROC observers.

    PubMed

    Khurd, Parmeshwar; Liu, Bin; Gindi, Gene

    2010-02-01

    Detection of multiple lesions in images is a medically important task and free-response receiver operating characteristic (FROC) analyses and its variants, such as alternative FROC (AFROC) analyses, are commonly used to quantify performance in such tasks. However, ideal observers that optimize FROC or AFROC performance metrics have not yet been formulated in the general case. If available, such ideal observers may turn out to be valuable for imaging system optimization and in the design of computer aided diagnosis techniques for lesion detection in medical images. In this paper, we derive ideal AFROC and FROC observers. They are ideal in that they maximize, amongst all decision strategies, the area, or any partial area, under the associated AFROC or FROC curve. Calculation of observer performance for these ideal observers is computationally quite complex. We can reduce this complexity by considering forms of these observers that use false positive reports derived from signal-absent images only. We also consider a Bayes risk analysis for the multiple-signal detection task with an appropriate definition of costs. A general decision strategy that minimizes Bayes risk is derived. With particular cost constraints, this general decision strategy reduces to the decision strategy associated with the ideal AFROC or FROC observer.

  12. Novel Functional Complexity of Polycystin-1 by GPS Cleavage In Vivo: Role in Polycystic Kidney Disease

    PubMed Central

    Kurbegovic, Almira; Kim, Hyunho; Xu, Hangxue; Yu, Shengqiang; Cruanès, Julie; Maser, Robin L.; Boletta, Alessandra; Trudel, Marie

    2014-01-01

    Polycystin-1 (Pc1) cleavage at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is required for normal kidney morphology in humans and mice. We found a complex pattern of endogenous Pc1 forms by GPS cleavage. GPS cleavage generates not only the heterodimeric cleaved full-length Pc1 (Pc1cFL) in which the N-terminal fragment (NTF) remains noncovalently associated with the C-terminal fragment (CTF) but also a novel (Pc1) form (Pc1deN) in which NTF becomes detached from CTF. Uncleaved Pc1 (Pc1U) resides primarily in the endoplasmic reticulum (ER), whereas both Pc1cFL and Pc1deN traffic through the secretory pathway in vivo. GPS cleavage is not a prerequisite, however, for Pc1 trafficking in vivo. Importantly, Pc1deN is predominantly found at the plasma membrane of renal epithelial cells. By functional genetic complementation with five Pkd1 mouse models, we discovered that CTF plays a crucial role in Pc1deN trafficking. Our studies support GPS cleavage as a critical regulatory mechanism of Pc1 biogenesis and trafficking for proper kidney development and homeostasis. PMID:24958103

  13. Cleavage strain in the Variscan fold belt, County Cork, Ireland, estimated from stretched arsenopyrite rosettes

    USGS Publications Warehouse

    Ford, M.; Ferguson, C.C.

    1985-01-01

    In south-west Ireland, hydrothermally formed arsenopyrite crystals in a Devonian mudstone have responded to Variscan deformation by brittle extension fracture and fragment separation. The interfragment gaps and terminal extension zones of each crystal are infilled with fibrous quartz. Stretches within the cleavage plane have been calculated by the various methods available, most of which can be modified to incorporate terminal extension zones. The Strain Reversal Method is the most accurate currently available but still gives a minimum estimate of the overall strain. The more direct Hossain method, which gives only slightly lower estimates with this data, is more practical for field use. A strain ellipse can be estimated from each crystal rosette composed of three laths (assuming the original interlimb angles were all 60??) and, because actual rather than relative stretches are estimated, this provides a lower bound to the area increase in the plane of cleavage. Based on the average of our calculated strain ellipses this area increase is at least 114% and implies an average shortening across the cleavage of at least 53%. However, several lines of evidence suggest that the cleavage deformation was more intense and more oblate than that calculated, and we argue that a 300% area increase in the cleavage plane and 75% shortening across the cleavage are more realistic estimates of the true strain. Furthermore, the along-strike elongation indicated is at least 80%, which may be regionally significant. Estimates of orogenic contraction derived from balanced section construction should therefore take into account the possibility of a substantial strike elongation, and tectonic models that can accommodate such elongations need to be developed. ?? 1985.

  14. Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

    PubMed Central

    Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

    2010-01-01

    BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

  15. Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2

    PubMed Central

    Reinke, Lennart Michel; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael

    2017-01-01

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated. PMID:28636671

  16. Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2.

    PubMed

    Reinke, Lennart Michel; Spiegel, Martin; Plegge, Teresa; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael; Pöhlmann, Stefan

    2017-01-01

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.

  17. Cleavage of amyloid precursor protein by an archaeal presenilin homologue PSH

    PubMed Central

    Dang, Shangyu; Wu, Shenjie; Wang, Jiawei; Li, Hongbo; Huang, Min; He, Wei; Li, Yue-Ming; Wong, Catherine C. L.; Shi, Yigong

    2015-01-01

    Aberrant cleavage of amyloid precursor protein (APP) by γ-secretase contributes to the development of Alzheimer’s disease. More than 200 disease-derived mutations have been identified in presenilin (the catalytic subunit of γ-secretase), making modulation of γ-secretase activity a potentially attractive therapeutic opportunity. Unfortunately, the technical challenges in dealing with intact γ-secretase have hindered discovery of modulators and demand a convenient substitute approach. Here we report that, similar to γ-secretase, the archaeal presenilin homolog PSH faithfully processes the substrate APP C99 into Aβ42, Aβ40, and Aβ38. The molar ratio of the cleavage products Aβ42 over Aβ40 by PSH is nearly identical to that by γ-secretase. The proteolytic activity of PSH is specifically suppressed by presenilin-specific inhibitors. Known modulators of γ-secretase also modulate PSH similarly in terms of the Aβ42/Aβ40 ratio. Structural analysis reveals association of a known γ-secretase inhibitor with PSH between its two catalytic aspartate residues. These findings identify PSH as a surrogate protease for the screening of agents that may regulate the protease activity and the cleavage preference of γ-secretase. PMID:25733893

  18. Chymase Cleavage of Stem Cell Factor Yields a Bioactive, Soluble Product

    NASA Astrophysics Data System (ADS)

    Longley, B. Jack; Tyrrell, Lynda; Ma, Yongsheng; Williams, David A.; Halaban, Ruth; Langley, Keith; Lu, Hsieng S.; Schechter, Norman M.

    1997-08-01

    Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

  19. Simple equations to simulate closed-loop recycling liquid-liquid chromatography: Ideal and non-ideal recycling models.

    PubMed

    Kostanyan, Artak E

    2015-12-04

    The ideal (the column outlet is directly connected to the column inlet) and non-ideal (includes the effects of extra-column dispersion) recycling equilibrium-cell models are used to simulate closed-loop recycling counter-current chromatography (CLR CCC). Simple chromatogram equations for the individual cycles and equations describing the transport and broadening of single peaks and complex chromatograms inside the recycling closed-loop column for ideal and non-ideal recycling models are presented. The extra-column dispersion is included in the theoretical analysis, by replacing the recycling system (connecting lines, pump and valving) by a cascade of Nec perfectly mixed cells. To evaluate extra-column contribution to band broadening, two limiting regimes of recycling are analyzed: plug-flow, Nec→∞, and maximum extra-column dispersion, Nec=1. Comparative analysis of ideal and non-ideal models has shown that when the volume of the recycling system is less than one percent of the column volume, the influence of the extra-column processes on the CLR CCC separation may be neglected. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

    PubMed Central

    Jablonski, Joseph; Clementz, Mark; Ryan, Kevin; Valente, Susana T.

    2014-01-01

    The 3’ end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages. Processing of the 3’ end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5’ cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3’ end processing of HIV-1 mRNAs. PMID:24835792

  1. Caspase-2 cleavage of tau reversibly impairs memory.

    PubMed

    Zhao, Xiaohui; Kotilinek, Linda A; Smith, Benjamin; Hlynialuk, Chris; Zahs, Kathleen; Ramsden, Martin; Cleary, James; Ashe, Karen H

    2016-11-01

    In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

  2. DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion

    PubMed Central

    Schwarz, Friedrich W.; van Aelst, Kara; Tóth, Júlia; Seidel, Ralf; Szczelkun, Mark D.

    2011-01-01

    DNA cleavage by the Type III Restriction–Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision. PMID:21724613

  3. AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

    PubMed

    Kobayashi, Maki; Aida, Masatoshi; Nagaoka, Hitoshi; Begum, Nasim A; Kitawaki, Yoko; Nakata, Mikiyo; Stanlie, Andre; Doi, Tomomitsu; Kato, Lucia; Okazaki, Il-mi; Shinkura, Reiko; Muramatsu, Masamichi; Kinoshita, Kazuo; Honjo, Tasuku

    2009-12-29

    To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

  4. Cathepsins limit macrophage necroptosis through cleavage of Rip1 kinase.

    PubMed

    McComb, Scott; Shutinoski, Bojan; Thurston, Susan; Cessford, Erin; Kumar, Kriti; Sad, Subash

    2014-06-15

    It has recently been shown that programmed necrosis, necroptosis, may play a key role in the development of inflammation. Deciphering the regulation of this pathway within immune cells may therefore have implications in pathology associated with inflammatory diseases. We show that treatment of macrophages with the pan caspase inhibitor (zVAD-FMK) results in both increased phosphorylation and decreased cleavage of receptor interacting protein kinase-1 (Rip1), leading to necroptosis that is dependent on autocrine TNF signaling. Stimulation of cells with TLR agonists such as LPS in the presence of zVAD-FMK also induced Rip1-phosphorylation via a TNFR-independent mechanism. Further examination of Rip1 expression under these stimulatory conditions revealed a regulatory cleavage of Rip1 in macrophages that is not apparently attributable to caspase-8. Instead, we provide novel evidence that cysteine family cathepsins, which are highly abundant in myeloid cells, can also cleave Rip1 kinase. Using small interfering RNA knockdown, specific cathepsin inhibitors, and cell-free cleavage assays, we demonstrate that cysteine cathepsins B and S can directly cleave Rip1. Finally, we demonstrate that only through combined inhibition of cathepsins and caspase-8 could a potent induction of macrophage necroptosis be achieved. These data reveal a novel mechanism of regulation of necroptosis by cathepsins within macrophage cells. Copyright © 2014 by The American Association of Immunologists, Inc.

  5. Surface Antigens Common to Mouse Cleavage Embryos and Primitive Teratocarcinoma Cells in Culture

    PubMed Central

    Artzt, Karen; Dubois, Philippe; Bennett, Dorothea; Condamine, Hubert; Babinet, Charles; Jacob, François

    1973-01-01

    Syngeneic antisera have been produced in mouse strain 129/Sv-CP males against the primitive cells of teratocarcinoma. These sera react specifically with the primitive cells and are negative on various types of differentiated teratoma cells derived from the same original tumor. They are negative on all other mouse cells tested, with the exception of male germ cells and cleavage-stage embryos. Thus, teratoma cells possess cell-surface antigens in common with normal cleavage-stage embryos. Images PMID:4355379

  6. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    PubMed

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

    Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

  7. A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44.

    PubMed

    Stoeck, Alexander; Keller, Sascha; Riedle, Svenja; Sanderson, Michael P; Runz, Steffen; Le Naour, Francois; Gutwein, Paul; Ludwig, Andreas; Rubinstein, Eric; Altevogt, Peter

    2006-02-01

    Ectodomain shedding is a proteolytic mechanism by which transmembrane molecules are converted into a soluble form. Cleavage is mediated by metalloproteases and proceeds in a constitutive or inducible fashion. Although believed to be a cell-surface event, there is increasing evidence that cleavage can take place in intracellular compartments. However, it is unknown how cleaved soluble molecules get access to the extracellular space. By analysing L1 (CD171) and CD44 in ovarian carcinoma cells, we show in the present paper that the cleavage induced by ionomycin, APMA (4-aminophenylmercuric acetate) or MCD (methyl-beta-cyclodextrin) is initiated in an endosomal compartment that is subsequently released in the form of exosomes. Calcium influx augmented the release of exosomes containing functionally active forms of ADAM10 (a disintegrin and metalloprotease 10) and ADAM17 [TACE (tumour necrosis factor a-converting enzyme)] as well as CD44 and L1 cytoplasmic cleavage fragments. Cleavage could also proceed in released exosomes, but only depletion of ADAM10 by small interfering RNA blocked cleavage under constitutive and induced conditions. In contrast, cleavage of L1 in response to PMA occurred at the cell surface and was mediated by ADAM17. We conclude that different ADAMs are involved in distinct cellular compartments and that ADAM10 is responsible for shedding in vesicles. Our findings open up the possibility that exosomes serve as a platform for ectodomain shedding and as a vehicle for the cellular export of soluble molecules.

  8. Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis.

    PubMed

    Pène, Véronique; Lemasson, Matthieu; Harper, Francis; Pierron, Gérard; Rosenberg, Arielle R

    2017-01-01

    In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before

  9. Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis

    PubMed Central

    Pène, Véronique; Lemasson, Matthieu; Harper, Francis; Pierron, Gérard; Rosenberg, Arielle R.

    2017-01-01

    In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before

  10. Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses.

    PubMed

    Eckhard, Ulrich; Huesgen, Pitter F; Schilling, Oliver; Bellac, Caroline L; Butler, Georgina S; Cox, Jennifer H; Dufour, Antoine; Goebeler, Verena; Kappelhoff, Reinhild; Keller, Ulrich Auf dem; Klein, Theo; Lange, Philipp F; Marino, Giada; Morrison, Charlotte J; Prudova, Anna; Rodriguez, David; Starr, Amanda E; Wang, Yili; Overall, Christopher M

    2016-01-01

    Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with

  11. Phosphodiester models for cleavage of nucleic acids

    PubMed Central

    2018-01-01

    Nucleic acids that store and transfer biological information are polymeric diesters of phosphoric acid. Cleavage of the phosphodiester linkages by protein enzymes, nucleases, is one of the underlying biological processes. The remarkable catalytic efficiency of nucleases, together with the ability of ribonucleic acids to serve sometimes as nucleases, has made the cleavage of phosphodiesters a subject of intensive mechanistic studies. In addition to studies of nucleases by pH-rate dependency, X-ray crystallography, amino acid/nucleotide substitution and computational approaches, experimental and theoretical studies with small molecular model compounds still play a role. With small molecules, the importance of various elementary processes, such as proton transfer and metal ion binding, for stabilization of transition states may be elucidated and systematic variation of the basicity of the entering or departing nucleophile enables determination of the position of the transition state on the reaction coordinate. Such data is important on analyzing enzyme mechanisms based on synergistic participation of several catalytic entities. Many nucleases are metalloenzymes and small molecular models offer an excellent tool to construct models for their catalytic centers. The present review tends to be an up to date summary of what has been achieved by mechanistic studies with small molecular phosphodiesters. PMID:29719577

  12. Mismatch cleavage by single-strand specific nucleases

    PubMed Central

    Till, Bradley J.; Burtner, Chris; Comai, Luca; Henikoff, Steven

    2004-01-01

    We have investigated the ability of single-strand specific (sss) nucleases from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single-nucleotide polymorphism analysis. The TILLING (Targeting Induced Local Lesions IN Genomes) mismatch cleavage protocol was used with the LI-COR gel detection system to assay cleavage of amplified heteroduplexes derived from a variety of induced mutations and naturally occurring polymorphisms. We found that purified nucleases derived from celery (CEL I), mung bean sprouts and Aspergillus (S1) were able to specifically cleave nearly all single base pair mismatches tested. Optimal nicking of heteroduplexes for mismatch detection was achieved using higher pH, temperature and divalent cation conditions than are routinely used for digestion of single-stranded DNA. Surprisingly, crude plant extracts performed as well as the highly purified preparations for this application. These observations suggest that diverse members of the S1 family of sss nucleases act similarly in cleaving non-specifically at bulges in heteroduplexes, and single-base mismatches are the least accessible because they present the smallest single-stranded region for enzyme binding. We conclude that a variety of sss nucleases and extracts can be effectively used for high-throughput mutation and polymorphism discovery. PMID:15141034

  13. Crystal structure of the 25 kDa subunit of human cleavage factor Im

    PubMed Central

    Coseno, Molly; Martin, Georges; Berger, Christopher; Gilmartin, Gregory; Keller, Walter; Doublié, Sylvie

    2008-01-01

    Cleavage factor Im is an essential component of the pre-messenger RNA 3′-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein–protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic α/β/α fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Å, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing. PMID:18445629

  14. A Python Analytical Pipeline to Identify Prohormone Precursors and Predict Prohormone Cleavage Sites

    PubMed Central

    Southey, Bruce R.; Sweedler, Jonathan V.; Rodriguez-Zas, Sandra L.

    2008-01-01

    Neuropeptides and hormones are signaling molecules that support cell–cell communication in the central nervous system. Experimentally characterizing neuropeptides requires significant efforts because of the complex and variable processing of prohormone precursor proteins into neuropeptides and hormones. We demonstrate the power and flexibility of the Python language to develop components of an bioinformatic analytical pipeline to identify precursors from genomic data and to predict cleavage as these precursors are en route to the final bioactive peptides. We identified 75 precursors in the rhesus genome, predicted cleavage sites using support vector machines and compared the rhesus predictions to putative assignments based on homology to human sequences. The correct classification rate of cleavage using the support vector machines was over 97% for both human and rhesus data sets. The functionality of Python has been important to develop and maintain NeuroPred (http://neuroproteomics.scs.uiuc.edu/neuropred.html), a user-centered web application for the neuroscience community that provides cleavage site prediction from a wide range of models, precision and accuracy statistics, post-translational modifications, and the molecular mass of potential peptides. The combined results illustrate the suitability of the Python language to implement an all-inclusive bioinformatics approach to predict neuropeptides that encompasses a large number of interdependent steps, from scanning genomes for precursor genes to identification of potential bioactive neuropeptides. PMID:19169350

  15. Development and application of bond cleavage reactions in bioorthogonal chemistry.

    PubMed

    Li, Jie; Chen, Peng R

    2016-03-01

    Bioorthogonal chemical reactions are a thriving area of chemical research in recent years as an unprecedented technique to dissect native biological processes through chemistry-enabled strategies. However, current concepts of bioorthogonal chemistry have largely centered on 'bond formation' reactions between two mutually reactive bioorthogonal handles. Recently, in a reverse strategy, a collection of 'bond cleavage' reactions has emerged with excellent biocompatibility. These reactions have expanded our bioorthogonal chemistry repertoire, enabling an array of exciting new biological applications that range from the chemically controlled spatial and temporal activation of intracellular proteins and small-molecule drugs to the direct manipulation of intact cells under physiological conditions. Here we highlight the development and applications of these bioorthogonal cleavage reactions. Furthermore, we lay out challenges and propose future directions along this appealing avenue of research.

  16. The Role of Character Strengths in Adolescent Romantic Relationships: An Initial Study on Partner Selection and Mates' Life Satisfaction

    ERIC Educational Resources Information Center

    Weber, Marco; Ruch, Willibald

    2012-01-01

    The present study investigated the role of 24 character strengths in 87 adolescent romantic relationships focusing on their role in partner selection and their role in mates' life satisfaction. Measures included the Values in Action Inventory of Strengths for Youth, the Students' Life Satisfaction Scale, and an Ideal Partner Profiler for the…

  17. Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha

    PubMed Central

    Diaz, Jason P.; Chirayil, Rachel; Chirayil, Sara; Tom, Martin; Head, Katie J.; Luebke, Kevin J.

    2014-01-01

    We have found a small molecule that specifically inhibits cleavage of a precursor to the oncogenic miRNA, miR-21, by the microprocessor complex of Drosha and DGCR8. We identified novel ligands for the apical loop of this precursor from a screen of 14,024 N-substituted oligoglycines (peptoids) in a microarray format. Eight distinct compounds with specific affinity were obtained, three having affinities for the targeted loop in the low micromolar range and greater than 15-fold discrimination against a closely related hairpin. One of these compounds completely inhibits microprocessor cleavage of a miR-21 primary transcript at concentrations at which cleavage of another miRNA primary transcript, pri-miR-16, is little affected. The apical loop of pri-miR-21, placed in the context of pri-miR-16, is sufficient for inhibition of microprocessor cleavage by the peptoid. This compound also inhibits cleavage of pri-miR-21 containing the pri-miR-16 apical loop, suggesting an additional site of association within pri-miR-21. The reported peptoid is the first example of a small molecule that inhibits microprocessor cleavage by binding to the apical loop of a pri-miRNA. PMID:24497550

  18. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

    PubMed Central

    Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

    2017-01-01

    Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

  19. cis-Apa: a practical linker for the microwave-assisted preparation of cyclic pseudopeptides via RCM cyclative cleavage.

    PubMed

    Baron, Alice; Verdié, Pascal; Martinez, Jean; Lamaty, Frédéric

    2011-02-04

    A new linker cis-5-aminopent-3-enoic acid (cis-Apa) was prepared for the synthesis of cyclic pseudopeptides by cyclization-cleavage by using ring-closing methatesis (RCM). We developed a new synthetic pathway for the preparation of the cis-Apa linker that was tested in the cyclization-cleavage process of different RGD peptide sequences. Different macrocyclic peptidomimetics were prepared by using this integrated microwave-assisted method, showing that the readily available cis-Apa amino acid is well adapted as a linker in the cyclization-cleavage process.

  20. Mining HIV protease cleavage data using genetic programming with a sum-product function.

    PubMed

    Yang, Zheng Rong; Dalby, Andrew R; Qiu, Jing

    2004-12-12

    In order to design effective HIV inhibitors, studying and understanding the mechanism of HIV protease cleavage specification is critical. Various methods have been developed to explore the specificity of HIV protease cleavage activity. However, success in both extracting discriminant rules and maintaining high prediction accuracy is still challenging. The earlier study had employed genetic programming with a min-max scoring function to extract discriminant rules with success. However, the decision will finally be degenerated to one residue making further improvement of the prediction accuracy difficult. The challenge of revising the min-max scoring function so as to improve the prediction accuracy motivated this study. This paper has designed a new scoring function called a sum-product function for extracting HIV protease cleavage discriminant rules using genetic programming methods. The experiments show that the new scoring function is superior to the min-max scoring function. The software package can be obtained by request to Dr Zheng Rong Yang.

  1. RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis.

    PubMed

    D'Avino, Pier Paolo; Savoian, Matthew S; Capalbo, Luisa; Glover, David M

    2006-11-01

    Several studies indicate that spindle microtubules determine the position of the cleavage plane at the end of cell division, but their exact role in triggering the formation and ingression of the cleavage furrow is still unclear. Here we show that in Drosophila depletion of either the GAP (GTPase-activating protein) or the kinesin-like subunit of the evolutionary conserved centralspindlin complex prevents furrowing without affecting the association of astral microtubules with the cell cortex. Moreover, time-lapse imaging indicates that astral microtubules serve to deliver the centralspindlin complex to the equatorial cortex just before furrow formation. However, when the GAP-signaling component was mislocalized around the entire cortex using a membrane-tethering motif, this caused ectopic furrowing even in the absence of its motor partner. Thus, the GAP component of centralspindlin is both necessary and sufficient for furrow formation and ingression and astral microtubules provide a route for its delivery to the cleavage site.

  2. Coupling fibroblast growth factor 23 production and cleavage: iron deficiency, rickets, and kidney disease.

    PubMed

    Wolf, Myles; White, Kenneth E

    2014-07-01

    High levels of fibroblast growth factor 23 (FGF23) cause the rare disorders of hypophosphatemic rickets and are a risk factor for cardiovascular disease and death in patients with chronic kidney disease (CKD). Despite major advances in understanding FGF23 biology, fundamental aspects of FGF23 regulation in health and in CKD remain mostly unknown. Autosomal dominant hypophosphatemic rickets (ADHR) is caused by gain-of-function mutations in FGF23 that prevent its proteolytic cleavage, but affected individuals experience a waxing and waning course of phosphate wasting. This led to the discovery that iron deficiency is an environmental trigger that stimulates FGF23 expression and hypophosphatemia in ADHR. Unlike osteocytes in ADHR, normal osteocytes couple increased FGF23 production with commensurately increased FGF23 cleavage to ensure that normal phosphate homeostasis is maintained in the event of iron deficiency. Simultaneous measurement of FGF23 by intact and C-terminal assays supported these breakthroughs by providing minimally invasive insight into FGF23 production and cleavage in bone. These findings also suggest a novel mechanism of FGF23 elevation in patients with CKD, who are often iron deficient and demonstrate increased FGF23 production and decreased FGF23 cleavage, consistent with an acquired state that mimics the molecular pathophysiology of ADHR. Iron deficiency stimulates FGF23 production, but normal osteocytes couple increased FGF23 production with increased cleavage to maintain normal circulating levels of biologically active hormone. These findings uncover a second level of FGF23 regulation within osteocytes, failure of which culminates in elevated levels of biologically active FGF23 in ADHR and perhaps CKD.

  3. Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots-peptide assembly.

    PubMed

    Wang, Jianhao; Fan, Jie; Liu, Li; Ding, Shumin; Liu, Xiaoqian; Wang, Jianpeng; Gao, Liqian; Chattopadhaya, Souvik; Miao, Peng; Xia, Jiang; Qiu, Lin; Jiang, Pengju

    2017-10-01

    Herein, a novel assay has been developed for monitoring PreScission protease (His-PSP) mediated enzyme cleavage of ATTO 590 labeled peptide substrate (ATTO-LEV). This novel method is based on combining the use of capillary electrophoresis and fluorescence detection (CE-FL) to dynamically monitor the enzyme cleavage activity. A multivalent peptide substrate was first constructed by immobilizing His-tagged ATTO 590 labeled peptide substrate (ATTO-LEVH6) onto the surface of CdSe/ZnS quantum dots (QDs). Once successfully immobilized, the novel multivalent peptide substrate resulted in the Förster resonance energy transfer (FRET) from QDs to ATTO 590. The ATTO-LEVH6-QD assembly was then incubated with His-PSP to study the proteolytic cleavage of surface bound ATTO-LEVH6 by CE-FL. Our data suggests that PreScission-mediated proteolytic cleavage is enzyme concentration- and incubation time-dependent. By combining capillary electrophoresis, QDs and FRET, our study herein not only provides a new method for the detection and dynamically monitoring of PSP enzyme cleavage activity, but also can be extended to the detection of many other enzymes and proteases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. A density functional theory study on peptide bond cleavage at aspartic residues: direct vs cyclic intermediate hydrolysis.

    PubMed

    Sang-aroon, Wichien; Amornkitbamrung, Vittaya; Ruangpornvisuti, Vithaya

    2013-12-01

    In this work, peptide bond cleavages at carboxy- and amino-sides of the aspartic residue in a peptide model via direct (concerted and step-wise) and cyclic intermediate hydrolysis reaction pathways were explored computationally. The energetics, thermodynamic properties, rate constants, and equilibrium constants of all hydrolysis reactions, as well as their energy profiles were computed at the B3LYP/6-311++G(d,p) level of theory. The result indicated that peptide bond cleavage of the Asp residue occurred most preferentially via the cyclic intermediate hydrolysis pathway. In all reaction pathways, cleavage of the peptide bond at the amino-side occurred less preferentially than at the carboxy-side. The overall reaction rate constants of peptide bond cleavage of the Asp residue at the carboxy-side for the assisted system were, in increasing order: concerted < step-wise < cyclic intermediate.

  5. The efficiency of dentin sialoprotein-phosphophoryn processing is affected by mutations both flanking and distant from the cleavage site.

    PubMed

    Yang, Robert T; Lim, Glendale L; Dong, Zhihong; Lee, Arthur M; Yee, Colin T; Fuller, Robert S; Ritchie, Helena H

    2013-02-22

    Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G(447)↓D(448) cleavage site in DSP-PP(240) had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P(4) to P(4)' blocked, impaired, or enhanced DSP-PP(240) cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP(240) had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP(240) significantly modified the amount of PP(240) product generated from DSP-PP(240) precursor protein cleavage suggests that such mutation may affect the mineralization process.

  6. Osteoporosis: Are we measuring what we intend to measure? In search of the ideal bone strength study

    NASA Astrophysics Data System (ADS)

    de Riese, Cornelia

    2006-02-01

    In 1991 the World Health Organization (WHO) defined osteoporosis as a "loss of bone mass and micro architectural deterioration of the skeleton leading to increased risk of fracture." 1,2 Since microarchitecture can not be measured directly, a panel of the WHO recommended that the diagnosis be made according to a quantifiable surrogate marker, calcium mineral, in bone. Subsequently in 1994, the definition focused on the actual bone "density," giving densitometric technology a central place in establishing the diagnosis of osteoporosis. 3,4 But soon it became obvious that there was only limited correlation between bone mineral density (BMD) and actual occurrence of fractures and that decreases in bone mass account for only about 50% of the deterioration of bone strength with aging. In other words only about 60% of bone strength is related to BMD. 5 Recent developments in bone research have shown that bone mineral density in itself is not sufficient to accurately predict fracture risk. Bone is composed of inorganic calcium apatite crystals that mineralize an organic type I collagen matrix. The degree of mineralization, the properties of the collagen matrix, crystal size, trabecular orientation, special distribution of the different components and many more factors are all impacting bone strength. 6-14 Human cadaver studies have confirmed the correlation between bone density and bone. 26 strength. 5,15-20 Changes in cancellous bone morphology appear to lead to a disproportionate decrease in bone strength. 21-26 When postmenopausal women are stratified by age, obvious differences between BMD and actual fracture risk are observed. 24 Felsenberg eloquently summarizes what he calls the "Bone Quality Framework." In great detail he talks about the geometry and micro- architecture of bone and how the different components are related to functional stability. 27 Are our current testing modalities appropriately addressing these structural factors? Are we keeping in mind that in

  7. Relationship of condom strength to failure during use.

    PubMed

    1980-10-01

    Less-than-ideal environmental conditions, especially in developing countries with tropical or desert climates, prolonged storage times because of unpredictable supply and distribution, and inexperience with warehousing and logistics causing haphazard turnover of stocks can accelerate deterioration of condoms and render them unsuitable for use. As condom strength standards have never been related directly to failure during use, a Program for the Introduction and Adaptation of Contraceptive Technology (PIACT) study, in collaboration with Planned Parenthood of Seattle-King County, Washington, was conducted to determine the actual relationship between condom strength and failure during use (see July 1980 issue of Contraception). The study found that: 1) air burst test parameters can effectively and sensitively measure changes in condom strength; 2) condoms produced by Western industrial standards exceed by a wide margin the minimum strength required for effective use; and 3) stored condoms should not necessarily be thrown out if they are uniform in strength, even though they fall below accepted standards for new condoms. The study also brought out the issue of condom packaging. The potent deteriorating effect of ultraviolet light on condoms is well-known, and it is therefore suggested that condoms be packaged in foil or opaque laminates on both sides. A separate study requested by the U.S. Agency for International Development investigating the relationship between the 2 tests for condom strength (air burst standards as used in the PIACT study and tensile strength measurements) showed that air burst data and tensile strength parameters closely reflected the same characteristics, thus providing support for the use of air burst strength measurements for predicting useful life of stored condoms.

  8. Effect of substrate RNA sequence on the cleavage reaction by a short ribozyme.

    PubMed Central

    Ohmichi, T; Okumoto, Y; Sugimoto, N

    1998-01-01

    Leadzyme is a ribozyme that requires Pb2+. The catalytic sequence, CUGGGAGUCC, binds to an RNA substrate, GGACC downward arrowGAGCCAG, cleaving the RNA substrate at one site. We have investigated the effect of the substrate sequence on the cleavage activity of leadzyme using mutant substrates in order to structurally understand the RNA catalysis. The results showed that leadzyme acted as a catalyst for single site cleavage of a C5 deletion mutant substrate, GGAC downward arrowGAGCCAG, as well as the wild-type substrate. However, a mutant substrate GGACCGACCAG, which had G8 deleted from the wild-type substrate, was not cleaved. Kinetic studies by surface plasmon resonance indicated that the difference between active and inactive structures reflected the slow association and dissociation rate constants of complex formation induced by Pb2+rather than differences in complex stability. CD spectra showed that the active form of the substrate-leadzyme complex was rearranged by Pb2+binding. The G8 of the wild-type substrate, which was absent in the inactive complex, is not near the cleavage site. Thus, these results show that the active substrate-leadzyme complex has a Pb2+binding site at the junction between the unpaired region (asymmetric internal loop) and the stem region, which is distal to the cleavage site. Pb2+may play a role in rearranging the bases in the asymmetric internal loop to the correct position for catalysis. PMID:9837996

  9. Preferential cleavage sites for Sau3A restriction endonuclease in human ribosomal DNA.

    PubMed

    Kupriyanova, N S; Kirilenko, P M; Netchvolodov, K K; Ryskov, A P

    2000-07-21

    Previous studies of cloned ribosomal DNA (rDNA) variants isolated from the cosmid library of human chromosome 13 have revealed some disproportion in representativity of different rDNA regions (N. S. Kupriyanova, K. K. Netchvolodov, P. M. Kirilenko, B. I. Kapanadze, N. K. Yankovsky, and A. P. Ryskov, Mol. Biol. 30, 51-60, 1996). Here we show nonrandom cleavage of human rDNA with Sau3A or its isoshizomer MboI under mild hydrolysis conditions. The hypersensitive cleavage sites were found to be located in the ribosomal intergenic spacer (rIGS), especially in the regions of about 5-5.5 and 11 kb upstream of the rRNA transcription start point. This finding is based on sequencing mapping of the rDNA insert ends in randomly selected cosmid clones of human chromosome 13 and on the data of digestion kinetics of cloned and noncloned human genomic rDNA with Sau3A and MboI. The results show that a methylation status and superhelicity state of the rIGS have no effect on cleavage site sensitivity. It is interesting that all primary cleavage sites are adjacent to or entering into Alu or Psi cdc 27 retroposons of the rIGS suggesting a possible role of neighboring sequences in nuclease accessibility. The results explain nonequal representation of rDNA sequences in the human genomic DNA library used for this study. Copyright 2000 Academic Press.

  10. Kirkwood–Buff integrals for ideal solutions

    PubMed Central

    Ploetz, Elizabeth A.; Bentenitis, Nikolaos; Smith, Paul E.

    2010-01-01

    The Kirkwood–Buff (KB) theory of solutions is a rigorous theory of solution mixtures which relates the molecular distributions between the solution components to the thermodynamic properties of the mixture. Ideal solutions represent a useful reference for understanding the properties of real solutions. Here, we derive expressions for the KB integrals, the central components of KB theory, in ideal solutions of any number of components corresponding to the three main concentration scales. The results are illustrated by use of molecular dynamics simulations for two binary solutions mixtures, benzene with toluene, and methanethiol with dimethylsulfide, which closely approach ideal behavior, and a binary mixture of benzene and methanol which is nonideal. Simulations of a quaternary mixture containing benzene, toluene, methanethiol, and dimethylsulfide suggest this system displays ideal behavior and that ideal behavior is not limited to mixtures containing a small number of components. PMID:20441282

  11. Effect of hot extrusion, other constituents, and temperature on the strength and fracture of polycrystalline MgO

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rice, R.W.

    Improved agreement was confirmed between the Petch intercept and single-crystal yield stresses at 22 C. Hot-extruded MgO crystal specimens stressed parallel with the resultant axial texture (1) gave the highest and least-scattered strength-grain size results at 22 C, (2) showed direct fractographic evidence of microplastic initiated fracture at 22 C and showed macroscopic yield at 1,315 and especially 1,540 C, and (3) fractured entirely via transgranular cleavage, except for intergranular failure initiation from one or a few grain boundary surfaces exposed on the subsequent fracture surface, mainly at 1,540 C. Hot-extruded, hot-pressed MgO billets gave comparable strength when fracture initiatedmore » transgranularly, but lower strength when fracture initiated from one or especially a few grain boundary surfaces exposed on the fracture. The extent and frequency of such boundary fracture increased with test temperature. While oxide additions of [<=] 5% or impurities in hot-pressed or hot-extruded MgO can make limited strength increases at larger grain sizes, those having limited solubility can limit strength at finer grain sizes, as can coarser surface finish. Overall, MgO strength is seen as a balance between flaw and microplastic controlled failure, with several parameters shifting the balance.« less

  12. Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: determinants and constraints.

    PubMed

    Lai, Zon W; Hanchapola, Iresha; Steer, David L; Smith, A Ian

    2011-06-14

    ADAM17, also known as tumor necrosis factor α-converting enzyme, is involved in the ectodomain shedding of many integral membrane proteins. We have previously reported that ADAM17 is able to mediate the cleavage secretion of the ectodomain of human angiotensin-converting enzyme 2 (ACE2), a functional receptor for the severe acute respiratory syndrome coronavirus. In this study, we demonstrate that purified recombinant human ADAM17 is able to cleave a 20-amino acid peptide mimetic corresponding to the extracellular juxtamembrane region of human ACE2 between Arg(708) and Ser(709). A series of peptide analogues were also synthesized, showing that glutamate subtitution at Arg(708) and/or Arg(710) attenuated the cleavage process, while alanine substitution at Arg(708) and/or Ser(709) did not inhibit peptide cleavage by recombinant ADAM17. Analysis of CD spectra showed a minimal difference in the secondary structure of the peptide analogues in the buffer system used for the ADAM17 cleavage assay. The observation of the shedding profiles of ACE2 mutants expressing CHO-K1 and CHO-P cells indicates that the Arg(708) → Glu(708) mutation and the Arg(708)Arg(710) → Glu(708)Glu(710) double mutation produced increases in the amount of ACE2 shed when stimulated by phorbol ester PMA. In summary, we have demonstrated that ADAM17 is able to cleave ACE2 peptide sequence analogues between Arg(708) and Ser(709). These findings also indicate that Arg(708) and Arg(710) play a role in site recognition in the regulation of ACE2 ectodomain shedding mediated by ADAM17.

  13. [Cleavage of DNA fragments induced by UV nanosecond laser excitation at 193 nm].

    PubMed

    Vtiurina, N N; Grokhovskiĭ, S L; Filimonov, I V; Medvedkov, O I; Nechipurenko, D Iu; Vasil'ev, S A; Nechipurenko, Iu D

    2011-01-01

    The cleavage of dsDNA fragments in aqueous solution after irradiation with UV laser pulses at 193 nm has been studied. Samples were investigated using polyacrylamide gel electrophoresis. The intensity of damage of particular phosphodiester bond after hot alkali treatment was shown to depend on the base pair sequence. It was established that the probability of cleavage is twice higher for sites of DNA containing two or more successively running guanine residues. A possible mechanism of damage to the DNA molecule connected with the migration of holes along the helix is discussed.

  14. Control of cleavage spindle orientation in Caenorhabditis elegans: The role of the genes par-2 and par-3

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cheng, N.N.; Kirby, C.M.; Kemphues, K.J.

    1995-02-01

    Polarized asymmetric divisions play important roles in the development of plants and animals. The first two embryonic cleavages of Caenorhabditis elegans provide an opportunity to study the mechanisms controlling polarized asymmetric divisions. The first cleavage is unequal, producing daughters with different sizes and fates. The daughter blastomeres divide with different orientations at the second cleavage; the anterior blastomere divides equally across the long axis of the egg, whereas the posterior blastomere divides unequally along the long axis. We report here the results of our analysis of the genes par-2 and par-3 with respect to their contribution to the polarity ofmore » these divisions. Strong loss-of-function mutations in both genes lead to an equal first cleavage and an altered second cleavage. Interestingly, the mutations exhibit striking gene-specific differences at the second cleavage. The par-2 mutations lead to transverse spindle orientations in both blastomeres, whereas par-3 mutations lead to longitudinal spindle orientations in both blastomeres. The spindle orientation defects correlate with defects in centrosome movements during both the first and the second cell cycle. Temperature shift experiments with par-2 (it5ts) indicate that the par-2(+) activity is not required after the two-cell stage. Analysis of double mutants shows that par-3 is epistatic to par-2. We propose a model wherein par-2(+) and par-3(+) act in concert during the first cell cycle to affect asymmetric modification of the cytoskeleton. This polar modification leads to different behaviors of centrosomes in the anterior and posterior and leads ultimately to blastomere-specific spindle orientations at the second cleavage. 44 refs., 5 figs., 5 tabs.« less

  15. Modeling Radial Holoblastic Cleavage: A Laboratory Activity for Developmental Biology.

    ERIC Educational Resources Information Center

    Ellis, Linda K.

    2000-01-01

    Introduces a laboratory activity designed for an undergraduate developmental biology course. Uses Play-Doh (plastic modeling clay) to build a multicellular embryo in order to provide a 3-D demonstration of cleavage. Includes notes for the instructor and student directions. (YDS)

  16. The Efficiency of Dentin Sialoprotein-Phosphophoryn Processing Is Affected by Mutations Both Flanking and Distant from the Cleavage Site*

    PubMed Central

    Yang, Robert T.; Lim, Glendale L.; Dong, Zhihong; Lee, Arthur M.; Yee, Colin T.; Fuller, Robert S.; Ritchie, Helena H.

    2013-01-01

    Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G447↓D448 cleavage site in DSP-PP240 had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P4 to P4′ blocked, impaired, or enhanced DSP-PP240 cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP240 had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP240 significantly modified the amount of PP240 product generated from DSP-PP240 precursor protein cleavage suggests that such mutation may affect the mineralization process. PMID:23297400

  17. Evaluation of fusion protein cleavage site sequences of Newcastle disease virus in genotype matched vaccines.

    PubMed

    Kim, Shin-Hee; Chen, Zongyan; Yoshida, Asuka; Paldurai, Anandan; Xiao, Sa; Samal, Siba K

    2017-01-01

    Newcastle disease virus (NDV) causes a devastating poultry disease worldwide. Frequent outbreaks of NDV in chickens vaccinated with conventional live vaccines suggest a need to develop new vaccines that are genetically matched against circulating NDV strains, such as the genotype V virulent strains currently circulating in Mexico and Central America. In this study, a reverse genetics system was developed for the virulent NDV strain Mexico/01/10 strain and used to generate highly attenuated vaccine candidates by individually modifying the cleavage site sequence of fusion (F) protein. The cleavage site sequence of parental virus was individually changed to those of the avirulent NDV strain LaSota and other serotypes of avian paramyxoviruses (APMV serotype-2, -3, -4, -6, -7, -8, and -9). In general, these mutations affected cell-to-cell fusion activity in vitro and the efficiency of the F protein cleavage and made recombinant Mexico/01/10 (rMex) virus highly attenuated in chickens. When chickens were immunized with the rMex mutant viruses and challenged with the virulent parent virus, there was reduced challenge virus shedding compared to birds immunized with the heterologous vaccine strain LaSota. Among the vaccine candidates, rMex containing the cleavage site sequence of APMV-2 induced the highest neutralizing antibody titer and completely protected chickens from challenge virus shedding. These results show the role of the F protein cleavage site sequence of each APMV type in generating genotype V-matched vaccines and the efficacy of matched vaccine strains to provide better protection against NDV strains currently circulating in Mexico.

  18. LBSizeCleav: improved support vector machine (SVM)-based prediction of Dicer cleavage sites using loop/bulge length.

    PubMed

    Bao, Yu; Hayashida, Morihiro; Akutsu, Tatsuya

    2016-11-25

    Dicer is necessary for the process of mature microRNA (miRNA) formation because the Dicer enzyme cleaves pre-miRNA correctly to generate miRNA with correct seed regions. Nonetheless, the mechanism underlying the selection of a Dicer cleavage site is still not fully understood. To date, several studies have been conducted to solve this problem, for example, a recent discovery indicates that the loop/bulge structure plays a central role in the selection of Dicer cleavage sites. In accordance with this breakthrough, a support vector machine (SVM)-based method called PHDCleav was developed to predict Dicer cleavage sites which outperforms other methods based on random forest and naive Bayes. PHDCleav, however, tests only whether a position in the shift window belongs to a loop/bulge structure. In this paper, we used the length of loop/bulge structures (in addition to their presence or absence) to develop an improved method, LBSizeCleav, for predicting Dicer cleavage sites. To evaluate our method, we used 810 empirically validated sequences of human pre-miRNAs and performed fivefold cross-validation. In both 5p and 3p arms of pre-miRNAs, LBSizeCleav showed greater prediction accuracy than PHDCleav did. This result suggests that the length of loop/bulge structures is useful for prediction of Dicer cleavage sites. We developed a novel algorithm for feature space mapping based on the length of a loop/bulge for predicting Dicer cleavage sites. The better performance of our method indicates the usefulness of the length of loop/bulge structures for such predictions.

  19. Characterization and Modeling of the Collision Induced Dissociation Patterns of Deprotonated Glycosphingolipids: Cleavage of the Glycosidic Bond

    NASA Astrophysics Data System (ADS)

    Rožman, Marko

    2016-01-01

    Glycosphingolipid fragmentation behavior was investigated by combining results from analysis of a series of negative ion tandem mass spectra and molecular modeling. Fragmentation patterns extracted from 75 tandem mass spectra of mainly acidic glycosphingolipid species (gangliosides) suggest prominent cleavage of the glycosidic bonds with retention of the glycosidic oxygen atom by the species formed from the reducing end (B and Y ion formation). Dominant product ions arise from dissociation of sialic acids glycosidic bonds whereas product ions resulting from cleavage of other glycosidic bonds are less abundant. Potential energy surfaces and unimolecular reaction rates of several low-energy fragmentation pathways leading to cleavage of glycosidic bonds were estimated in order to explain observed dissociation patterns. Glycosidic bond cleavage in both neutral (unsubstituted glycosyl group) and acidic glycosphingolipids was the outcome of the charge-directed intramolecular nucleophilic substitution (SN2) mechanism. According to the suggested mechanism, the nucleophile in a form of carboxylate or oxyanion attacks the carbon at position one of the sugar ring, simultaneously breaking the glycosidic bond and yielding an epoxide. For gangliosides, unimolecular reaction rates suggest that dominant product ions related to the cleavage of sialic acid glycosidic bonds are formed via direct dissociation channels. On the other hand, low abundant product ions related to the dissociation of other glycosidic bonds are more likely to be the result of sequential dissociation. Although results from this study mainly contribute to the understanding of glycosphingolipid fragmentation chemistry, some mechanistic findings regarding cleavage of the glycosidic bond may be applicable to other glycoconjugates.

  20. Nanorelief of the natural cleavage surface of triglycine sulphate crystals with substitutional and interstitial impurities

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Belugina, N. V.; Gainutdinov, R. V.; Tolstikhina, A. L., E-mail: alla@ns.crys.ras.ru

    2011-11-15

    Ferroelectric triglycine sulphate crystals (TGS) with substitutional (LADTGS+ADP, DTGS) and interstitial (Cr) impurities have been studied by atomic-force microscopy, X-ray diffraction, and X-ray fluorescence. The nanorelief parameters of the mirror cleavage TGS(010) surface have been measured with a high accuracy. A correlation between the crystal defect density in the bulk and the cleavage surface nanorelief is revealed at the submicrometer level.

  1. High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

    PubMed

    Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

    2015-06-17

    High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This

  2. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    PubMed Central

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2015-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

  3. Ideal regularization for learning kernels from labels.

    PubMed

    Pan, Binbin; Lai, Jianhuang; Shen, Lixin

    2014-08-01

    In this paper, we propose a new form of regularization that is able to utilize the label information of a data set for learning kernels. The proposed regularization, referred to as ideal regularization, is a linear function of the kernel matrix to be learned. The ideal regularization allows us to develop efficient algorithms to exploit labels. Three applications of the ideal regularization are considered. Firstly, we use the ideal regularization to incorporate the labels into a standard kernel, making the resulting kernel more appropriate for learning tasks. Next, we employ the ideal regularization to learn a data-dependent kernel matrix from an initial kernel matrix (which contains prior similarity information, geometric structures, and labels of the data). Finally, we incorporate the ideal regularization to some state-of-the-art kernel learning problems. With this regularization, these learning problems can be formulated as simpler ones which permit more efficient solvers. Empirical results show that the ideal regularization exploits the labels effectively and efficiently. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Intuitionistic fuzzy n-fold KU-ideal of KU-algebra

    NASA Astrophysics Data System (ADS)

    Mostafa, Samy M.; Kareem, Fatema F.

    2018-05-01

    In this paper, we apply the notion of intuitionistic fuzzy n-fold KU-ideal of KU-algebra. Some types of ideals such as intuitionistic fuzzy KU-ideal, intuitionistic fuzzy closed ideal and intuitionistic fuzzy n-fold KU-ideal are studied. Also, the relations between intuitionistic fuzzy n-fold KU-ideal and intuitionistic fuzzy KU-ideal are discussed. Furthermore, a few results of intuitionistic fuzzy n-fold KU-ideals of a KU-algebra under homomorphism are discussed.

  5. Examples for Non-Ideal Solution Thermodynamics Study

    ERIC Educational Resources Information Center

    David, Carl W.

    2004-01-01

    A mathematical model of a non-ideal solution is presented, where it is shown how and where the non-ideality manifests itself in the standard thermodynamics tableau. Examples related to the non-ideal solution thermodynamics study are also included.

  6. Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes

    PubMed Central

    Sheng, Gang; Gogakos, Tasos; Wang, Jiuyu; Zhao, Hongtu; Serganov, Artem; Juranek, Stefan

    2017-01-01

    Abstract We have undertaken a systematic structural study of Thermus thermophilus Argonaute (TtAgo) ternary complexes containing single-base bulges positioned either within the seed segment of the guide or target strands and at the cleavage site. Our studies establish that single-base bulges 7T8, 5A6 and 4A5 on the guide strand are stacked-into the duplex, with conformational changes localized to the bulge site, thereby having minimal impact on the cleavage site. By contrast, single-base bulges 6’U7’ and 6’A7’ on the target strand are looped-out of the duplex, with the resulting conformational transitions shifting the cleavable phosphate by one step. We observe a stable alignment for the looped-out 6’N7’ bulge base, which stacks on the unpaired first base of the guide strand, with the looped-out alignment facilitated by weakened Watson–Crick and reversed non-canonical flanking pairs. These structural studies are complemented by cleavage assays that independently monitor the impact of bulges on TtAgo-mediated cleavage reaction. PMID:28911094

  7. Ideal Magnetic Dipole Scattering

    NASA Astrophysics Data System (ADS)

    Feng, Tianhua; Xu, Yi; Zhang, Wei; Miroshnichenko, Andrey E.

    2017-04-01

    We introduce the concept of tunable ideal magnetic dipole scattering, where a nonmagnetic nanoparticle scatters light as a pure magnetic dipole. High refractive index subwavelength nanoparticles usually support both electric and magnetic dipole responses. Thus, to achieve ideal magnetic dipole scattering one has to suppress the electric dipole response. Such a possibility was recently demonstrated for the so-called anapole mode, which is associated with zero electric dipole scattering. By spectrally overlapping the magnetic dipole resonance with the anapole mode, we achieve ideal magnetic dipole scattering in the far field with tunable strong scattering resonances in the near infrared spectrum. We demonstrate that such a condition can be realized at least for two subwavelength geometries. One of them is a core-shell nanosphere consisting of a Au core and silicon shell. It can be also achieved in other geometries, including nanodisks, which are compatible with current nanofabrication technology.

  8. Quercetin-Iron Complex: Synthesis, Characterization, Antioxidant, DNA Binding, DNA Cleavage, and Antibacterial Activity Studies.

    PubMed

    Raza, Aun; Xu, Xiuquan; Xia, Li; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-11-01

    Quercetin-iron (II) complex was synthesized and characterized by elemental analysis, ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, mass spectrometry, proton nuclear magnetic resonance spectroscopy, thermogravimetry and differential scanning calorimetry, scanning electron micrography and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal:ligand) of the complex. Antioxidant study of the quercetin and its metal complex against 2, 2-di-phenyl-1-picryl hydrazyl radical showed that the complex has much more radical scavenging activity than free quercetin. The interaction of quercetin-iron (II) complex with DNA was determined using ultraviolet visible spectra, fluorescence spectra and agarose gel electrophoresis. The results showed that quercetin-iron (II) complex can intercalate moderately with DNA, quench a strong intercalator ethidium bromide and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form to nicked circular form and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was an oxidative cleavage pathway. These results revealed the potential nuclease activity of complex to cleave DNA. In addition, antibacterial activity of complex on E.coli and S. aureus was also investigated. The results showed that complex has higher antibacterial activity than ligand.

  9. Metabolic Engineering to Develop a Pathway for the Selective Cleavage of Carbon-Nitrogen Bonds

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    John J. Kilbane II

    The objective of the project is to develop a biochemical pathway for the selective cleavage of C-N bonds in molecules found in petroleum. Specifically a novel biochemical pathway will be developed for the selective cleavage of C-N bonds in carbazole. The cleavage of the first C-N bond in carbazole is accomplished by the enzyme carbazole dioxygenase, that catalyzes the conversion of carbazole to 2-aminobiphenyl-2,3-diol. The genes encoding carbazole dioxygenase were cloned from Sphingomonas sp. GTIN11 and from Pseudomonas resinovorans CA10. The selective cleavage of the second C-N bond has been challenging, and efforts to overcome that challenge have been themore » focus of recent research in this project. Enrichment culture experiments succeeded in isolating bacterial cultures that can metabolize 2-aminobiphenyl, but no enzyme capable of selectively cleaving the C-N bond in 2-aminobiphenyl has been identified. Aniline is very similar to the structure of 2-aminobiphenyl and aniline dioxygenase catalyzes the conversion of aniline to catechol and ammonia. For the remainder of the project the emphasis of research will be to simultaneously express the genes for carbazole dioxygenase and for aniline dioxygenase in the same bacterial host and then to select for derivative cultures capable of using carbazole as the sole source of nitrogen.« less

  10. Crystal Structure of the 25 kDa Subunit of Human Cleavage Factor I{m}

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Coseno,M.; Martin, G.; Berger, C.

    Cleavage factor Im is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic {alpha}/{beta}/{alpha} fold and a conserved catalytic sequence or Nudix box. We present heremore » the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Angstroms, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.« less

  11. Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

    PubMed

    Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark

    2018-05-29

    CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Effect of Mucoprotein on the Bond Strength of Resin Composite to Human Dentin

    PubMed Central

    Pinzon, Lilliam M; Powers, John M; O'Keefe, Kathy; Dusevish, Vladimir; Spencer, Paulette; Marshall, Grayson W

    2010-01-01

    The purpose of this study was to test the bond strength and analyze the morphology of the dentin-adhesive interface of two etch and rinse and two self-etch adhesive systems with two kinds of artificial saliva (with and without 450 mg/L mucin) contamination under different conditions of decontaminating the interface. Bonded specimens were sectioned perpendicularly to the bonded surface in 1-mm thick slabs. These 1-mm thick slabs were remounted in acrylic blocks and sectioned in sticks perpendicular to the bonding interfaces with a 1-mm2 area. Nine specimens from each condition were tested after 24 hours on a testing machine (Instron) at a speed of 0.5 mm/min for a total of 360 specimens. Means and standard deviations of bond strength (MPa) were calculated. ANOVA showed significant differences as well as Fisher's PLSD intervals (p<0.05). Different groups results ranges: Control group 34-60 MPa, saliva without mucin 0-52 MPa, and saliva with mucin 0-57 MPa. Failure sites were mixed, adhesive failure was common for the low bond strength results. P&BNT with ideal conditions and following the manufacturer's instructions (control) had the highest bond strengths and the dentin-adhesive interface exhibited an ideal morphology of a etch and rinse system. SEM gave complementary visual evidence of the effect in the dentin/adhesive interface structure with some contaminated conditions compared to their respective control groups. This in-vitro artificial saliva model with and without mucin showed that an organic component of saliva could increase or decrease the bond strength depending on the specific bonding agent and decontamination procedure. PMID:14505182

  13. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    NASA Astrophysics Data System (ADS)

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

  14. The poly(A)-binding protein nuclear 1 suppresses alternative cleavage and polyadenylation sites.

    PubMed

    Jenal, Mathias; Elkon, Ran; Loayza-Puch, Fabricio; van Haaften, Gijs; Kühn, Uwe; Menzies, Fiona M; Oude Vrielink, Joachim A F; Bos, Arnold J; Drost, Jarno; Rooijers, Koos; Rubinsztein, David C; Agami, Reuven

    2012-04-27

    Alternative cleavage and polyadenylation (APA) is emerging as an important layer of gene regulation. Factors controlling APA are largely unknown. We developed a reporter-based RNAi screen for APA and identified PABPN1 as a regulator of this process. Genome-wide analysis of APA in human cells showed that loss of PABPN1 resulted in extensive 3' untranslated region shortening. Messenger RNA transcription, stability analyses, and in vitro cleavage assays indicated enhanced usage of proximal cleavage sites (CSs) as the underlying mechanism. Using Cyclin D1 as a test case, we demonstrated that enhanced usage of proximal CSs compromises microRNA-mediated repression. Triplet-repeat expansion in PABPN1 (trePABPN1) causes autosomal-dominant oculopharyngeal muscular dystrophy (OPMD). The expression of trePABPN1 in both a mouse model of OPMD and human cells elicited broad induction of proximal CS usage, linked to binding to endogenous PABPN1 and its sequestration in nuclear aggregates. Our results elucidate a novel function for PABPN1 as a suppressor of APA. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. The development of high strength corrosion resistant precipitation hardening cast steels

    NASA Astrophysics Data System (ADS)

    Abrahams, Rachel A.

    , give poor estimates of secondary phases in PHCSS. No measureable retained austenite was observed in any of the CB7Cu-1 steels studied, in spite of the fact that austenite is predicted by the constitution diagrams. A designed experiment using computationally derived phase equilibrium diagrams and actual experimental tests on CB7Cu of different compositions suggests that the ferrite phase is less stable than the constitution diagrams for austenitic stainless steels suggest. Delta ferrite was also more stable in slower-cooled sand cast material as compared to thin, fast-cooled investment cast material. High temperature solutionizing treatments were effective in dissolving delta ferrite at temperatures above 1900°F (˜1040°C). Delta ferrite dissolution was found to proceed at high rates during initial dissolution, and then was found to slow after 1 hour. Diffusion during the later stages is well-predicted by classical diffusion models. Repeated solution treatments were found to modestly increase both ductility and strength, likely due to subgrain refinement through austenite regrowth. Multistaged aging provided superior strength and toughness increases over similarly peak-aged and near peak-aged material aged at a single temperature. Peak-aged material fractography suggested that low energy quasi-cleavage fracture was likely due to age precipitate embrittlement along with some nucleation of MnS particulates at prior austenite grain boundaries. Yield strengths approaching 190 ksi (1310MPa) can be achieved in CB7Cu-1 if appropriate best-practices "+" processing techniques are used. This includes hot isostatic processing to reduce solidification segregation and heal microporosity, high temperature homogenization for effective age hardening and ferrite reduction, double-cycle solutionizing for structure refinement, and multistaged age strengthening for finer precipitate control. The experimental prototype 11-11PH (Fe-Ni-Cr-Ti-Mo) casting alloys was cast and was found to be

  16. piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

    PubMed

    Goh, Wee Siong Sho; Falciatori, Ilaria; Tam, Oliver H; Burgess, Ralph; Meikar, Oliver; Kotaja, Noora; Hammell, Molly; Hannon, Gregory J

    2015-05-15

    MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells. © 2015 Goh et al.; Published by Cold Spring Harbor Laboratory Press.

  17. Active Site Mutations Change the Cleavage Specificity of Neprilysin

    PubMed Central

    Sexton, Travis; Hitchcook, Lisa J.; Rodgers, David W.; Bradley, Luke H.; Hersh, Louis B.

    2012-01-01

    Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe563 and Ser546. Among the mutants studied in detail we observed changes in their activity towards leucine5-enkephalin, insulin B chain, and amyloid β1–40. For example, NEPF563I displayed an increase in preference towards cleaving leucine5-enkephalin relative to insulin B chain, while mutant NEPS546E was less discriminating than neprilysin. Mutants NEPF563L and NEPS546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß1–40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential. PMID:22384224

  18. Is nucleophilic cleavage chemistry practical for 4-membered heterocycles?

    PubMed

    Banks, Harold D

    2009-11-07

    A computational study at the MP2(Full)/6-311++G(d,p)//MP2(Full)/6-31+G(d) level of the ammonolysis of halogen substituted azetidines, oxetanes and thietanes was performed in the gas phase and in the commonly used solvent, acetonitrile. Using the free energy of activation of a benchmark reaction for evaluation of synthetic viability, several haloazetidines and oxetanes that possessed the required reactivity were identified; however, no substituted thietane investigated herein was determined to be synthetically useful under the mild conditions selected for this study. In the case of the azetidines, the side reaction of displacement of halide ion was determined to be the preferred reaction course in acetonitrile; however, the amino product of the reactions of the 2-haloazetidines cleaved at an acceptable rate under mild conditions. For the oxetane derivatives investigated, 2-fluorooxetane proved to be a direct source of ring cleavage product. Nucleophilic cleavage of halogen-substituted azetidines and oxetanes is predicted to be a viable source of functionalized three-carbon moieties under mild conditions in organic synthesis.

  19. Effects of Olive Metabolites on DNA Cleavage Mediated by Human Type II Topoisomerases

    PubMed Central

    2016-01-01

    Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα. An extract from Phillyrea latifolia L., a member of the olive tree family, displayed high activity against the human enzyme. On the basis of previous metabolomics studies, we identified several polyphenols (hydroxytyrosol, oleuropein, verbascoside, tyrosol, and caffeic acid) as potential candidates for topoisomerase II poisons. Of these, hydroxytyrosol, oleuropein, and verbascoside enhanced topoisomerase II-mediated DNA cleavage. The potency of these olive metabolites increased 10–100-fold in the presence of an oxidant. Hydroxytyrosol, oleuropein, and verbascoside displayed hallmark characteristics of covalent topoisomerase II poisons. (1) The activity of the metabolites was abrogated by a reducing agent. (2) Compounds inhibited topoisomerase II activity when they were incubated with the enzyme prior to the addition of DNA. (3) Compounds were unable to poison a topoisomerase IIα construct that lacked the N-terminal domain. Because hydroxytyrosol, oleuropein, and verbascoside are broadly distributed across the olive family, extracts from the leaves, bark, and fruit of 11 olive tree species were tested for activity against human topoisomerase IIα. Several of the extracts enhanced enzyme-mediated DNA cleavage. Finally, a commercial olive leaf supplement and extra virgin olive oils pressed from a variety of Olea europea subspecies enhanced DNA cleavage mediated by topoisomerase IIα. Thus, olive metabolites appear to act as topoisomerase II poisons in complex formulations intended for human dietary consumption. PMID:26132160

  20. Crack Arrest Toughness of Two High Strength Steels (AISI 4140 and AISI 4340)

    NASA Astrophysics Data System (ADS)

    Ripling, E. J.; Mulherin, J. H.; Crosley, P. B.

    1982-04-01

    The crack initiation toughness ( K c ) and crack arrest toughness ( K a ) of AISI 4140 and AISI 4340 steel were measured over a range of yield strengths from 965 to 1240 MPa, and a range of test temperatures from -53 to +74°C. Emphasis was placed on K a testing since these values are thought to represent the minimum toughness of the steel as a function of loading rate. At the same yield strengths and test temperatures, K a for the AISI 4340 was about twice as high as it was for the AISI 4140. In addition, the K a values showed a more pronounced transition temperature than the K c values, when the data were plotted as a function of test temperature. The transition appeared to be associated with a change in fracture mechanism from cleavage to dimpled rupture as the test temperature was increased. The occurrence of a “pop-in” behavior at supertransition temperatures has not been found in lower strength steels, and its evaluation in these high strength steels was possible only because they are not especially tough at their supertransition temperatures. There is an upper toughness limit at which pop-in will not occur, and this was found for the AISI 4340 steel when it was tempered to its lowest yield strength (965 MPa). All the crack arrest data were identified as plane strain values, while only about one-half of the initiation values could be classified this way.

  1. Observation of Early Cleavage in Animal Development: A Simple Technique for Obtaining the Eggs of Rhabditis (Nematoda)

    ERIC Educational Resources Information Center

    Hinchliffe, J. R.

    1973-01-01

    Outlines the advantages of using the readily available eggs of the nematode Rhabditis in studying the early cleavage stages of animal development. Discusses the identification and life history of Rhabditis, how to culture and examine the organism, the cleavage stages and cell lineage, and sources of visual aids. (JR)

  2. Oxidative cleavage of diverse ethers by an extracellular fungal peroxygenase

    Treesearch

    Matthias Kinne; Marzena Poraj-Kobielska; Sally A. Ralph; Rene Ullrich; Martin Hofrichter; Kenneth E. Hammel

    2009-01-01

    Many litter-decay fungi secrete heme-thiolate peroxygenases that oxidize various organic chemicals, but little is known about the role or mechanism of these enzymes. We found that the extracellular peroxygenase of Agrocybe aegerita catalyzed the H2O2-dependent cleavage of environmentally significant...

  3. Improved Classification of Mammograms Following Idealized Training

    PubMed Central

    Hornsby, Adam N.; Love, Bradley C.

    2014-01-01

    People often make decisions by stochastically retrieving a small set of relevant memories. This limited retrieval implies that human performance can be improved by training on idealized category distributions (Giguère & Love, 2013). Here, we evaluate whether the benefits of idealized training extend to categorization of real-world stimuli, namely classifying mammograms as normal or tumorous. Participants in the idealized condition were trained exclusively on items that, according to a norming study, were relatively unambiguous. Participants in the actual condition were trained on a representative range of items. Despite being exclusively trained on easy items, idealized-condition participants were more accurate than those in the actual condition when tested on a range of item types. However, idealized participants experienced difficulties when test items were very dissimilar from training cases. The benefits of idealization, attributable to reducing noise arising from cognitive limitations in memory retrieval, suggest ways to improve real-world decision making. PMID:24955325

  4. Improved Classification of Mammograms Following Idealized Training.

    PubMed

    Hornsby, Adam N; Love, Bradley C

    2014-06-01

    People often make decisions by stochastically retrieving a small set of relevant memories. This limited retrieval implies that human performance can be improved by training on idealized category distributions (Giguère & Love, 2013). Here, we evaluate whether the benefits of idealized training extend to categorization of real-world stimuli, namely classifying mammograms as normal or tumorous. Participants in the idealized condition were trained exclusively on items that, according to a norming study, were relatively unambiguous. Participants in the actual condition were trained on a representative range of items. Despite being exclusively trained on easy items, idealized-condition participants were more accurate than those in the actual condition when tested on a range of item types. However, idealized participants experienced difficulties when test items were very dissimilar from training cases. The benefits of idealization, attributable to reducing noise arising from cognitive limitations in memory retrieval, suggest ways to improve real-world decision making.

  5. The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages

    NASA Astrophysics Data System (ADS)

    Peng, Zhou; Bu, Jiexun; McLuckey, Scott A.

    2017-09-01

    We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. [Figure not available: see fulltext.

  6. Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

    NASA Astrophysics Data System (ADS)

    Foreman, David J.; Dziekonski, Eric T.; McLuckey, Scott A.

    2018-04-01

    A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. [Figure not available: see fulltext.

  7. Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins.

    PubMed

    Foreman, David J; Dziekonski, Eric T; McLuckey, Scott A

    2018-04-30

    A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. Graphical Abstract ᅟ.

  8. ATP-Dependent C–F Bond Cleavage Allows the Complete Degradation of 4-Fluoroaromatics without Oxygen

    PubMed Central

    Tiedt, Oliver; Mergelsberg, Mario; Boll, Kerstin; Müller, Michael; Adrian, Lorenz; Jehmlich, Nico; von Bergen, Martin

    2016-01-01

    ABSTRACT Complete biodegradation of the abundant and persistent fluoroaromatics requires enzymatic cleavage of an arylic C–F bond, probably the most stable single bond of a biodegradable organic molecule. While in aerobic microorganisms defluorination of fluoroaromatics is initiated by oxygenases, arylic C–F bond cleavage has never been observed in the absence of oxygen. Here, an oxygen-independent enzymatic aryl fluoride bond cleavage is described during the complete degradation of 4-fluorobenzoate or 4-fluorotoluene to CO2 and HF in the denitrifying Thauera aromatica: the ATP-dependent defluorination of 4-fluorobenzoyl-coenzyme A (4-F-BzCoA) to benzoyl-coenzyme A (BzCoA) and HF, catalyzed by class I BzCoA reductase (BCR). Adaptation to growth with the fluoroaromatics was accomplished by the downregulation of a promiscuous benzoate-CoA ligase and the concomitant upregulation of 4-F-BzCoA-defluorinating/dearomatizing BCR on the transcriptional level. We propose an unprecedented mechanism for reductive arylic C–F bond cleavage via a Birch reduction-like mechanism resulting in a formal nucleophilic aromatic substitution. In the proposed anionic 4-fluorodienoyl-CoA transition state, fluoride elimination to BzCoA is favored over protonation to a fluorinated cyclic dienoyl-CoA. PMID:27507824

  9. Cleavage of transmembrane junction proteins and their role in regulating epithelial homeostasis

    PubMed Central

    Nava, Porfirio; Kamekura, Ryuta; Nusrat, Asma

    2013-01-01

    Epithelial tissues form a selective barrier that separates the external environment from the internal tissue milieu. Single epithelial cells are densely packed and associate via distinct intercellular junctions. Intercellular junction proteins not only control barrier properties of the epithelium but also play an important role in regulating epithelial homeostasis that encompasses cell proliferation, migration, differentiation and regulated shedding. Recent studies have revealed that several proteases target epithelial junction proteins during physiological maturation as well as in pathologic states such as inflammation and cancer. This review discusses mechanisms and biological consequences of transmembrane junction protein cleavage. The influence of junction protein cleavage products on pathogenesis of inflammation and cancer is discussed. PMID:24665393

  10. The interfacial strength of carbon nanofiber epoxy composite using single fiber pullout experiments.

    PubMed

    Manoharan, M P; Sharma, A; Desai, A V; Haque, M A; Bakis, C E; Wang, K W

    2009-07-22

    Carbon nanotubes and nanofibers are extensively researched as reinforcing agents in nanocomposites for their multifunctionality, light weight and high strength. However, it is the interface between the nanofiber and the matrix that dictates the overall properties of the nanocomposite. The current trend is to measure elastic properties of the bulk nanocomposite and then compare them with theoretical models to extract the information on the interfacial strength. The ideal experiment is single fiber pullout from the matrix because it directly measures the interfacial strength. However, the technique is difficult to apply to nanocomposites because of the small size of the fibers and the requirement for high resolution force and displacement sensing. We present an experimental technique for measuring the interfacial strength of nanofiber-reinforced composites using the single fiber pullout technique and demonstrate the technique for a carbon nanofiber-reinforced epoxy composite. The experiment is performed in situ in a scanning electron microscope and the interfacial strength for the epoxy composite was measured to be 170 MPa.

  11. An ideal free-kick

    NASA Astrophysics Data System (ADS)

    De Luca, R.; Faella, O.

    2017-01-01

    The kinematics of a free-kick is studied. As in projectile motion, the free-kick is ideal since we assume that a point-like ball moves in the absence of air resistance. We have experienced the fortunate conjuncture of a classical mechanics lecture taught right before an important football game. These types of sports events might trigger a great deal of attention from the classroom. The idealized problem is devised in such a way that students are eager to come to the end of the whole story.

  12. Impact of PCOS on early embryo cleavage kinetics.

    PubMed

    Wissing, M L; Bjerge, M R; Olesen, A I G; Hoest, T; Mikkelsen, A L

    2014-04-01

    This study investigated whether polycystic ovary syndrome (PCOS) affected early embryo development assessed by time-lapse analysis of embryo kinetics from fertilization to the blastocyst stage. This was a prospective cohort study of two pronuclei (2PN) embryos from 25 hyperandrogenic PCOS patients (110 2PN embryos), 26 normoandrogenic PCOS patients (140 2PN embryos) and 20 healthy, regularly cycling women (controls, 97 2PN embryos). Patients underwent the same baseline evaluation and the same ovarian stimulation from April 2010 to February 2013. Oocytes were fertilized by intracytoplasmic sperm injection and incubated in an EmbryoScope with pictures taken every 20 min in seven focal planes. Time to 2PN breakdown, first cleavage and cleavage to 3, 4, 5, 6, 7 and 8 cells, morula and blastocyst (t₂, t₃, t₄, t₅, t₆, t₇, t₈, t(M), t(B)) were annotated. Differences in embryo kinetics between groups were assessed by mixed modelling. Compared with controls, embryos from hyperandrogenic PCOS patients were significantly delayed at 2PN breakdown, t₂, t₃, t₄ and t₇ but not at t₅, t₆, t₈, t(M) or t(B). Embryos from hyperandrogenic PCOS women had developed slower from fertilization to the 8-cell stage compared with embryos from controls. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  13. As a Matter of Force—Systematic Biases in Idealized Turbulence Simulations

    NASA Astrophysics Data System (ADS)

    Grete, Philipp; O’Shea, Brian W.; Beckwith, Kris

    2018-05-01

    Many astrophysical systems encompass very large dynamical ranges in space and time, which are not accessible by direct numerical simulations. Thus, idealized subvolumes are often used to study small-scale effects including the dynamics of turbulence. These turbulent boxes require an artificial driving in order to mimic energy injection from large-scale processes. In this Letter, we show and quantify how the autocorrelation time of the driving and its normalization systematically change the properties of an isothermal compressible magnetohydrodynamic flow in the sub- and supersonic regime and affect astrophysical observations such as Faraday rotation. For example, we find that δ-in-time forcing with a constant energy injection leads to a steeper slope in kinetic energy spectrum and less-efficient small-scale dynamo action. In general, we show that shorter autocorrelation times require more power in the acceleration field, which results in more power in compressive modes that weaken the anticorrelation between density and magnetic field strength. Thus, derived observables, such as the line-of-sight (LOS) magnetic field from rotation measures, are systematically biased by the driving mechanism. We argue that δ-in-time forcing is unrealistic and numerically unresolved, and conclude that special care needs to be taken in interpreting observational results based on the use of idealized simulations.

  14. On controlling nonlinear dissipation in high order filter methods for ideal and non-ideal MHD

    NASA Technical Reports Server (NTRS)

    Yee, H. C.; Sjogreen, B.

    2004-01-01

    The newly developed adaptive numerical dissipation control in spatially high order filter schemes for the compressible Euler and Navier-Stokes equations has been recently extended to the ideal and non-ideal magnetohydrodynamics (MHD) equations. These filter schemes are applicable to complex unsteady MHD high-speed shock/shear/turbulence problems. They also provide a natural and efficient way for the minimization of Div(B) numerical error. The adaptive numerical dissipation mechanism consists of automatic detection of different flow features as distinct sensors to signal the appropriate type and amount of numerical dissipation/filter where needed and leave the rest of the region free from numerical dissipation contamination. The numerical dissipation considered consists of high order linear dissipation for the suppression of high frequency oscillation and the nonlinear dissipative portion of high-resolution shock-capturing methods for discontinuity capturing. The applicable nonlinear dissipative portion of high-resolution shock-capturing methods is very general. The objective of this paper is to investigate the performance of three commonly used types of nonlinear numerical dissipation for both the ideal and non-ideal MHD.

  15. Developmental bias in cleavage-stage mouse blastomeres

    PubMed Central

    Tabansky, Inna; Lenarcic, Alan; Draft, Ryan W.; Loulier, Karine; Keskin, Derin B; Rosains, Jacqueline; Rivera-Feliciano, José; Lichtman, Jeff W.; Livet, Jean; Stern, Joel NH; Sanes, Joshua R.; Eggan, Kevin

    2012-01-01

    Summary Introduction The cleavage stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, as preimplantation genetic diagnosis (PGD) requires removal of blastomeres from the early human embryo. To determine if blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, non-invasive combinatorial fluorescent labeling method for embryonic lineage tracing. Results When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into post-implantation stages, and therefore has relevance for subsequent development. Discussion The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues. PMID:23177476

  16. The association between global self-esteem, physical self-concept and actual vs ideal body size rating in Chinese primary school children.

    PubMed

    Lau, P W C; Lee, A; Ransdell, L; Yu, C W; Sung, R Y T

    2004-02-01

    To investigate whether the discrepancy between actual and ideal body size rating is related to Chinese children's global self-esteem and global physical self-concept. A cross-sectional study of school children who completed questionnaires related to global self-esteem, global physical self-concept, and actual vs ideal body size. A total of 386 Chinese children (44% girls and 56% boys) aged 7-13 y from a primary school in Hong Kong, China. Global self-esteem and physical self-concept were measured using the physical self-descriptive questionnaire. Actual vs ideal body size discrepancy was established using the silhouette matching task. No significant relationship was found between global self-esteem and actual-ideal body size discrepancy of children. Global physical self-concept had a moderate negative correlation (r=-0.12) with the body size discrepancy score and the discrepancy score explained very limited variance (R(2)=0.015; F(1, 296)=4.51; P<0.05) in global physical self-concept. Three body size discrepancy groups (none, positive, and negative) were examined to see if there were any significant differences in global self-esteem, global physical self-concept, and specific dimensions of physical self-concept. A significant overall difference was found between groups for global physical self-concept (F=3.73, P<0.05) and the physical self-concept subscales of physical activity (F=3.25, P<0.05), body fat (F=61.26, P<0.001), and strength (F=5.26, P<0.01). Boys scored significantly higher than girls on global physical self-concept-especially in the sport competence, strength, and endurance subscales. This study revealed that the actual-ideal body size discrepancy rating of Chinese children was not predictive of global physical self-concept and global self-esteem. These findings are contrary to those reported in Western children, which may mean that culture plays a role in the formation of body attitude.

  17. Environment-dependent interfacial strength using first principles thermodynamics: The example of the Pt-HfO2 interface

    NASA Astrophysics Data System (ADS)

    Cardona Quintero, Y.; Ramanath, Ganpati; Ramprasad, R.

    2013-10-01

    A parameter-free, quantitative, first-principles methodology to determine the environment-dependent interfacial strength of metal-metal oxide interfaces is presented. This approach uses the notion of the weakest link to identify the most likely cleavage plane, and first principles thermodynamics to calculate the average work of separation as a function of the environment (in this case, temperature and oxygen pressure). The method is applied to the case of the Pt-HfO2 interface, and it is shown that the computed environment-dependent work of separation is in quantitative agreement with available experimental data.

  18. Recharging Our Sense of Idealism: Concluding Thoughts

    ERIC Educational Resources Information Center

    D'Andrea, Michael; Dollarhide, Colette T.

    2011-01-01

    In this article, the authors aim to recharge one's sense of idealism. They argue that idealism is the Vitamin C that sustains one's commitment to implementing humanistic principles and social justice practices in the work of counselors and educators. The idealism that characterizes counselors and educators who are humanistic and social justice…

  19. Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs.

    PubMed

    Oka, M T; Arai, T; Hamaguchi, Y

    1990-12-01

    Two monoclonal antibodies against alpha-tubulin (YL1/2 and D2D6) were microinjected into the egg of the sand dollar Clypeaster japonicus, and their effects on cleavage of the egg were investigated. They had already been shown by immunoblotting to react specifically with egg tubulin and by immunofluorescence to stain the mitotic apparatus [OKA et al., (1990). Cell Motil. Cytoskel. 16:239-250]. Injection of YL1/2 prevented chromosome movement and cleavage, although the cleavage furrow developed in some cases. In all eggs injected at prometaphase, metaphase, or anaphase, the birefringence of the mitotic apparatus disappeared immediately after injection. Injection of D2D6 had no significant effect on mitosis or cleavage of whole eggs injected after nuclear disappearance, although it prevented the disappearance of the nuclear envelope in 54% of the eggs injected before the disappearance. FITC-conjugated D2D6 did not accumulate in the spindle when injected into the dividing sand dollar egg. These results indicate that YL1/2 disassembled microtubules, whereas D2D6 did not bind to microtubules in the living cell.

  20. Activation and reactivation of the RNA polymerase II trigger loop for intrinsic RNA cleavage and catalysis

    PubMed Central

    Čabart, Pavel; Jin, Huiyan; Li, Liangtao; Kaplan, Craig D

    2014-01-01

    In addition to RNA synthesis, multisubunit RNA polymerases (msRNAPs) support enzymatic reactions such as intrinsic transcript cleavage. msRNAP active sites from different species appear to exhibit differential intrinsic transcript cleavage efficiency and have likely evolved to allow fine-tuning of the transcription process. Here we show that a single amino-acid substitution in the trigger loop (TL) of Saccharomyces RNAP II, Rpb1 H1085Y, engenders a gain of intrinsic cleavage activity where the substituted tyrosine appears to participate in acid-base chemistry at alkaline pH for both intrinsic cleavage and nucleotidyl transfer. We extensively characterize this TL substitution for each of these reactions by examining the responses RNAP II enzymes to catalytic metals, altered pH, and factor inputs. We demonstrate that TFIIF stimulation of the first phosphodiester bond formation by RNAP II requires wild type TL function and that H1085Y substitution within the TL compromises or alters RNAP II responsiveness to both TFIIB and TFIIF. Finally, Mn2+ stimulation of H1085Y RNAP II reveals possible allosteric effects of TFIIB on the active center and cooperation between TFIIB and TFIIF. PMID:25764335

  1. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

    PubMed

    Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

  2. Mechanistic Analysis of Oxidative C–H Cleavages Using Inter- and Intramolecular Kinetic Isotope Effects

    PubMed Central

    Jung, Hyung Hoon; Floreancig, Paul E.

    2009-01-01

    A series of monodeuterated benzylic and allylic ethers were subjected to oxidative carbon–hydrogen bond cleavage to determine the impact of structural variation on intramolecular kinetic isotope effects in DDQ-mediated cyclization reactions. These values are compared to the corresponding intermolecular kinetic isotope effects that were accessed through subjecting mixtures of non-deuterated and dideuterated substrates to the reaction conditions. The results indicate that carbon–hydrogen bond cleavage is rate determining and that a radical cation is most likely a key intermediate in the reaction mechanism. PMID:20640173

  3. Cleavage of the Drosophila seminal protein Acp36DE in mated females enhances its sperm storage activity.

    PubMed

    Avila, Frank W; Wolfner, Mariana F

    2017-08-01

    Sperm storage in the mated female reproductive tract (RT) is required for optimal fertility in numerous species with internal fertilization. In Drosophila melanogaster, sperm storage is dependent on female receipt of seminal fluid proteins (SFPs) during mating. The seminal fluid protein Acp36DE is necessary for the accumulation of sperm into storage. In the female RT, Acp36DE localizes to the anterior mating plug and also to a site in the common oviduct, potentially "corralling" sperm near the entry sites into the storage organs. Genetic studies showed that Acp36DE is also required for a series of conformational changes of the uterus that begin at the onset of mating and are hypothesized to move sperm towards the entry sites of the sperm storage organs. After Acp36DE is transferred to the female RT, the protein is cleaved by the astacin-metalloprotease Semp1. However, the effect of this cleavage on Acp36DE's function in sperm accumulation into storage is unknown. We used mass spectrometry to identify the single cleavage site in Acp36DE. We then mutated this site and tested the effects on sperm storage. Mutations of Acp36DE's cleavage site that slowed or prevented cleavage of the protein slowed the accumulation of sperm into storage, although they did not affect uterine conformational changes in mated females. Moreover, the N-terminal cleavage product of Acp36DE was sufficient to mediate sperm accumulation in storage, and it did so faster than versions of Acp36DE that could not be cleaved or were only cleaved slowly. These results suggest that cleavage of Acp36E may increase the number of bioactive molecules within the female RT, a mechanism similar to that hypothesized for Semp1's other substrate, the seminal fluid protein ovulin. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. The in vitro cleavage of the hAtg proteins by cell death proteases.

    PubMed

    Norman, Joanna M; Cohen, Gerald M; Bampton, Edward T W

    2010-11-01

    It is becoming increasingly clear that there is crosstalk between the apoptotic and autophagic pathways, with autophagy helping to contribute to cell death by providing energy to allow the energy-requiring programmed cell death process to complete, as well as degrading cellular material in its own right. Recent evidence has suggested that Atg proteins can themselves be targets of caspases, providing potential regulation of autophagy as well as uncovering novel functions for fragments derived from Atg proteins. However, to date there has not been a detailed examination of which Atg proteins may be the targets of which death proteases. We show that the majority of human Atg (hAtg) proteins can be cleaved by calpain 1, which is activated in some apoptotic paradigms, as well as other forms of death. We also show that hAtg3 is cleaved by caspases-3, -6 and -8, hAtg6 (Beclin 1) is cleaved by caspase-3 and -6, while hAtg9, hAtg7 and the hAtg4 homologues can be cleaved by caspase-3. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL, along with cleavage of Atg3 and Atg4C. There were subtle effects of caspase inhibition on GFP-LC3 lipidation but more marked effects on the formation of GFP-LC3 puncta (a marker of autophagosome formation) and p62 degradation, indicating that caspase cleavage of autophagy-related proteins can affect the autophagic process. Notably we show that p62 is a target for caspase-6 and -8 cleavage.

  5. A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

    PubMed Central

    Lott, William B.; Pontius, Brian W.; von Hippel, Peter H.

    1998-01-01

    Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 μM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and >50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and >37.5 μM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2′-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5′-oxygen in the transition state. We suggest structural reasons why the Mg2+–La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction. PMID:9435228

  6. Familial Alzheimer’s mutations within APPTM increase Aβ42 production by enhancing accessibility of ɛ-cleavage site

    NASA Astrophysics Data System (ADS)

    Chen, Wen; Gamache, Eric; Rosenman, David J.; Xie, Jian; Lopez, Maria M.; Li, Yue-Ming; Wang, Chunyu

    2014-01-01

    The high Aβ42/Aβ40 production ratio is a hallmark of familial Alzheimer’s disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aβ is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ɛ-cleavage at either T48 or L49, resulting in subsequent production of Aβ42 or Aβ40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ɛ-cleavage, and consequently shift cleavage preference towards Aβ42.

  7. TRPC6 specifically interacts with APP to inhibit its cleavage by γ-secretase and reduce Aβ production

    PubMed Central

    Wang, Junfeng; Lu, Rui; Yang, Jian; Li, Hongyu; He, Zhuohao; Jing, Naihe; Wang, Xiaomin; Wang, Yizheng

    2015-01-01

    Generation of β-amyloid (Aβ) peptide in Alzheimer's disease involves cleavage of amyloid precursor protein (APP) by γ-secretase, a protease known to cleave several substrates, including Notch. Finding specific modulators for γ-secretase could be a potential avenue to treat the disease. Here, we report that transient receptor potential canonical (TRPC) 6 specifically interacts with APP leading to inhibition of its cleavage by γ-secretase and reduction in Aβ production. TRPC6 interacts with APP (C99), but not with Notch, and prevents C99 interaction with presenilin 1 (PS1). A fusion peptide derived from TRPC6 also reduces Aβ levels without effect on Notch cleavage. Crossing APP/PS1 mice with TRPC6 transgenic mice leads to a marked reduction in both plaque load and Aβ levels, and improvement in structural and behavioural impairment. Thus, TRPC6 specifically modulates γ-secretase cleavage of APP and preventing APP (C99) interaction with PS1 via TRPC6 could be a novel strategy to reduce Aβ formation. PMID:26581893

  8. Prenatal Organophosphates Exposure Alternates the Cleavage Plane Orientation of Apical Neural Progenitor in Developing Neocortex

    PubMed Central

    Chen, Xiao-Ping; Chen, Wei-Feng; Wang, Da-Wei

    2014-01-01

    Prenatal organophosphate exposure elicits long-term brain cytoarchitecture and cognitive function impairments, but the mechanism underlying the onset and development of neural progenitors remain largely unclear. Using precise positioned brain slices, we observed an alternated cleavage plane bias that emerged in the mitotic neural progenitors of embryonal neocortex with diazinion (DZN) and chlorpyrifos (CPF) pretreatment. In comparison with the control, DZN and CPF treatment induced decrease of vertical orientation, increase of oblique orientation, and increase of horizontal orientation. That is, the cleavage plane orientation bias had been rotated from vertical to horizontal after DZN and CPF treatment. Meanwhile, general morphology and mitotic index of the progenitors were unchanged. Acephate (ACP), another common organophosphate, had no significant effects on the cleavage plane orientation, cell morphology and mitotic index. These results represent direct evidence for the toxicity mechanism in onset multiplication of neural progenitors. PMID:24740262

  9. A peptide-based approach to evaluate the adaptability of influenza A virus to humans based on its hemagglutinin proteolytic cleavage site

    PubMed Central

    Straus, Marco R.; Whittaker, Gary R.

    2017-01-01

    Cleavage activation of the hemagglutinin (HA) protein by host proteases is a crucial step in the infection process of influenza A viruses (IAV). However, IAV exists in eighteen different HA subtypes in nature and their cleavage sites vary considerably. There is uncertainty regarding which specific proteases activate a given HA in the human respiratory tract. Understanding the relationship between different HA subtypes and human-specific proteases will be valuable in assessing the pandemic potential of circulating viruses. Here we utilized fluorogenic peptides mimicking the HA cleavage motif of representative IAV strains causing disease in humans or of zoonotic/pandemic potential and tested them with a range of proteases known to be present in the human respiratory tract. Our results show that peptides from the H1, H2 and H3 subtypes are cleaved efficiently by a wide range of proteases including trypsin, matriptase, human airway tryptase (HAT), kallikrein-related peptidases 5 (KLK5) and 12 (KLK12) and plasmin. Regarding IAVs currently of concern for human adaptation, cleavage site peptides from H10 viruses showed very limited cleavage by respiratory tract proteases. Peptide mimics from H6 viruses showed broader cleavage by respiratory tract proteases, while H5, H7 and H9 subtypes showed variable cleavage; particularly matriptase appeared to be a key protease capable of activating IAVs. We also tested HA substrate specificity of Factor Xa, a protease required for HA cleavage in chicken embryos and relevant for influenza virus production in eggs. Overall our data provide novel tool allowing the assessment of human adaptation of IAV HA subtypes. PMID:28358853

  10. Heterolytic Cleavage of H2 by Bifunctional Manganese(I) Complexes: Impact of Ligand Dynamics, Electrophilicity, and Base Positioning

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hulley, Elliott B.; Helm, Monte L.; Bullock, R. Morris

    2014-12-01

    We report the synthesis, characterization, and reactivity with H2 of a series of MnI complexes of the type [(P-P)Mn(L2)CO]+ (L2 = dppm, bppm, or (CO)2; P-P = PPhNMePPh or PPh2 NBn2 ) that bear pendant amine ligands designed to function as proton relays. The pendant amine was found to function as a hemilabile ligand; its binding strength is strongly affected by the ancillary ligand environment around Mn. Tuning the electrophilicity of the Mn center leads to systems capable of reversible heterolytic cleavage of the H-H bond. The strength of pendant amine binding can be balanced to protect the Mn centermore » while still leading to facile reactivity with H2. Neutral amine-bearing MnIH species were found to react with one-electron oxidants and, after proton and electron transfer reactions, regenerate MnI cationic species. The reactivity presented herein indicate that the Mn complexes we have developed are a promising platform for Mn-based H2 oxidation electrocatalyst development. The research was supported as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences. Pacific Northwest National Laboratory is operated by Battelle for DOE.« less

  11. On the accuracy of Whitham's method. [for steady ideal gas flow past cones

    NASA Technical Reports Server (NTRS)

    Zahalak, G. I.; Myers, M. K.

    1974-01-01

    The steady flow of an ideal gas past a conical body is studied by the method of matched asymptotic expansions and by Whitham's method in order to assess the accuracy of the latter. It is found that while Whitham's method does not yield a correct asymptotic representation of the perturbation field to second order in regions where the flow ahead of the Mach cone of the apex is disturbed, it does correctly predict the changes of the second-order perturbation quantities across a shock (the first-order shock strength). The results of the analysis are illustrated by a special case of a flat, rectangular plate at incidence.

  12. Genetic and environmental influences on thin-ideal internalization.

    PubMed

    Suisman, Jessica L; O'Connor, Shannon M; Sperry, Steffanie; Thompson, J Kevin; Keel, Pamela K; Burt, S Alexandra; Neale, Michael; Boker, Steven; Sisk, Cheryl; Klump, Kelly L

    2012-12-01

    Current research on the etiology of thin-ideal internalization focuses on psychosocial influences (e.g., media exposure). The possibility that genetic influences also account for variance in thin-ideal internalization has never been directly examined. This study used a twin design to estimate genetic effects on thin-ideal internalization and examine if environmental influences are primarily shared or nonshared in origin. Participants were 343 postpubertal female twins (ages: 12-22 years; M = 17.61) from the Michigan State University Twin Registry. Thin-ideal internalization was assessed using the Sociocultural Attitudes toward Appearance Questionnaire-3. Twin modeling suggested significant additive genetic and nonshared environmental influences on thin-ideal internalization. Shared environmental influences were small and non-significant. Although prior research focused on psychosocial factors, genetic influences on thin-ideal internalization were significant and moderate in magnitude. Research is needed to investigate possible interplay between genetic and nonshared environmental factors in the development of thin-ideal internalization. Copyright © 2012 Wiley Periodicals, Inc.

  13. RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage.

    PubMed

    Schaefer, Matthias; Pollex, Tim; Hanna, Katharina; Tuorto, Francesca; Meusburger, Madeleine; Helm, Mark; Lyko, Frank

    2010-08-01

    Dnmt2 proteins are the most conserved members of the DNA methyltransferase enzyme family, but their substrate specificity and biological functions have been a subject of controversy. We show here that, in addition to tRNA(Asp-GTC), tRNA(Val-AAC) and tRNA(Gly-GCC) are also methylated by Dnmt2. Drosophila Dnmt2 mutants showed reduced viability under stress conditions, and Dnmt2 relocalized to stress granules following heat shock. Strikingly, stress-induced cleavage of tRNAs was Dnmt2-dependent, and Dnmt2-mediated methylation protected tRNAs against ribonuclease cleavage. These results uncover a novel biological function of Dnmt2-mediated tRNA methylation, and suggest a role for Dnmt2 enzymes during the biogenesis of tRNA-derived small RNAs.

  14. Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin.

    PubMed

    Bergeron, Éric; Zivcec, Marko; Chakrabarti, Ayan K; Nichol, Stuart T; Albariño, César G; Spiropoulou, Christina F

    2015-05-01

    Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV's high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor's maturation to GP38, and Gn precursor's maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public

  15. Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin

    PubMed Central

    Bergeron, Éric; Zivcec, Marko; Chakrabarti, Ayan K.; Nichol, Stuart T.; Albariño, César G.; Spiropoulou, Christina F.

    2015-01-01

    Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV’s high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor’s maturation to GP38, and Gn precursor’s maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public

  16. Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

    PubMed Central

    Subramanian, Shoba; Hardt, Markus; Choe, Youngchool; Niles, Richard K.; Johansen, Eric B.; Legac, Jennifer; Gut, Jiri; Kerr, Iain D.; Craik, Charles S.; Rosenthal, Philip J.

    2009-01-01

    The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P1 – P4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P2 position. Second, with overlapping peptides spanning α and β globin and proteolysis-dependent 18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents. PMID:19357776

  17. Family Life and Developmental Idealism in Yazd, Iran

    PubMed Central

    Abbasi-Shavazi, Mohammad Jalal; Askari-Nodoushan, Abbas

    2012-01-01

    BACKGROUND This paper is motivated by the theory that developmental idealism has been disseminated globally and has become an international force for family and demographic change. Developmental idealism is a set of cultural beliefs and values about development and how development relates to family and demographic behavior. It holds that modern societies are causal forces producing modern families, that modern families help to produce modern societies, and that modern family change is to be expected. OBJECTIVE We examine the extent to which developmental idealism has been disseminated in Iran. We also investigate predictors of the dissemination of developmental idealism. METHODS We use survey data collected in 2007 from a sample of women in Yazd, a city in Iran. We examine the distribution of developmental idealism in the sample and the multivariate predictors of developmental idealism. RESULTS We find considerable support for the expectation that many elements of developmental idealism have been widely disseminated. Statistically significant majorities associate development with particular family attributes, believe that development causes change in families, believe that fertility reductions and age-at-marriage increases help foster development, and perceive family trends in Iran headed toward modernity. As predicted, parental education, respondent education, and income affect adherence to developmental idealism. CONCLUSIONS Developmental idealism has been widely disseminated in Yazd, Iran and is related to social and demographic factors in predicted ways. COMMENTS Although our data come from only one city, we expect that developmental idealism has been widely distributed in Iran, with important implications for family and demographic behavior. PMID:22942772

  18. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    PubMed

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-04

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  19. The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

    PubMed Central

    Pettit, Steven C.; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H.

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation. PMID:12477841

  20. Ideal Cardiovascular Health and Incident Cardiovascular Events

    PubMed Central

    Ommerborn, Mark J.; Blackshear, Chad T.; Hickson, DeMarc A.; Griswold, Michael E.; Kwatra, Japneet; Djousse, Luc; Clark, Cheryl R.

    2016-01-01

    Introduction The epidemiology of American Heart Association ideal cardiovascular health (CVH) metrics has not been fully examined in African Americans. This study examines associations of CVH metrics with incident cardiovascular disease (CVD) in the Jackson Heart Study, a longitudinal cohort study of CVD in African Americans. Methods Jackson Heart Study participants without CVD (N=4,702) were followed prospectively between 2000 and 2011. Incidence rates and Cox proportional hazard ratios estimated risks for incident CVD (myocardial infarction, stroke, cardiac procedures, and CVD mortality) associated with seven CVH metrics by sex. Analyses were performed in 2015. Results Participants were followed for a median 8.3 years; none had ideal health on all seven CVH metrics. The prevalence of ideal health was low for nutrition, physical activity, BMI, and blood pressure metrics. The age-adjusted CVD incidence rate (IR) per 1,000 person years was highest for individuals with the least ideal health metrics: zero to one (IR=12.5, 95% CI=9.7, 16.1), two (IR=8.2, 95% CI=6.5, 10.4), three (IR=5.7, 95% CI=4.2, 7.6), and four or more (IR=3.4, 95% CI=2.0, 5.9). Adjusting for covariates, individuals with four or more ideal CVH metrics had lower risks of incident CVD compared with those with zero or one ideal CVH metric (hazard ratio, 0.29; 95% CI=0.17, 0.52; p<0.001). Conclusions African Americans with more ideal CVH metrics have lower risks of incident CVD. Comprehensive preventive behavioral and clinical supports should be intensified to improve CVD risk for African Americans with few ideal CVH metrics. PMID:27539974

  1. Metallurgical/Alloy Optimization of High Strength and Wear Resistant Structural Quench and Tempered Steels

    NASA Astrophysics Data System (ADS)

    Stalheim, Douglas G.; Peimao, Fu; Linhao, Gu; Yongqing, Zhang

    Structural steels with yield strength requirements greater or equal to 690 MPa can be produced through controlled recrystallization hot rolling coupled with precipitation strengthening or purposeful heat treatment through quench and tempering (Q&T). High strength structural steel and wear/abrasion resistant requirements greater or equal to 360 Brinell hardness (BHN) are produced by the development of microstructures of tempered lower bainite and/or martensite through the Q&T process. While these Q&T microstructures can produce very high strengths and hardness levels making them ideal for 690 MPa plus yield strength or wear/abrasion resistant applications, they lack toughness/ductility and hence are very brittle and prone to cracking. While tempering the microstructures helps in improving the toughness/ductility and reducing the brittleness, strength and hardness can be sacrificed. In addition, these steels typically consist of alloy designs containing boron with carbon equivalents (CE) greater than 0.50 to achieve the desired microstructures. The higher CE has a negative influence on weldability.

  2. Observations of cleavage steps, slip traces and dislocation hollow cores on cleaved ?100? faces of ?-arginine phosphate monohydrate single crystals by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Sangwal, K.; Torrent-Burgués, J.; Sanz, F.; Servat, J.

    1997-03-01

    The results of an atomic force microscopy study of the nature of cleavage steps, observation of slip traces and formation of hollow cores at the centres of dislocations on the {100} faces of L-arginine phosphate monohydrate (LAP) single crystals grown from aqueous solutions are described and discussed. It was observed that: (1) most of the cleavage steps and all the slip traces are of elementary height, a = 1.085 nm; (2) the origin of a cleavage step may or may not have a hollow core; and (3) close to its origin, the curvature of a cleavage step may be positive or negative or may change from positive to negative. The results suggest that slip traces observed on the cleaved surfaces of LAP are formed during the cleavage process while the rounding and the rearrangement of elementary cleavage steps take place immediately after the occurrence of cleavage. Analysis of the results also shows that the dislocations responsible for the origin of hollow cores always represent a stress field state corresponding to a trapped solution of different local interface supersaturations.

  3. Novel insights into the fungal oxidation of monoaromatic and biarylic environmental pollutants by characterization of two new ring cleavage enzymes.

    PubMed

    Schlüter, Rabea; Lippmann, Ramona; Hammer, Elke; Gesell Salazar, Manuela; Schauer, Frieder

    2013-06-01

    The phenol-degrading yeast Trichosporon mucoides can oxidize and detoxify biarylic environmental pollutants such as dibenzofuran, diphenyl ether and biphenyl by ring cleavage. The degradation pathways are well investigated, but the enzymes involved are not. The high similarity of hydroxylated biphenyl derivatives and phenol raised the question if the enzymes of the phenol degradation are involved in ring cleavage or whether specific enzymes are necessary. Purification of enzymes from T. mucoides with catechol cleavage activity demonstrated the existence of three different enzymes: a classical catechol-1,2-dioxygenase (CDO), not able to cleave the aromatic ring system of 3,4-dihydroxybiphenyl, and two novel enzymes with a high affinity towards 3,4-dihydroxybiphenyl. The comparison of the biochemical characteristics and mass spectrometric sequence data of these three enzymes demonstrated that they have different substrate specificities. CDO catalyzes the ortho-cleavage of dihydroxylated monoaromatic compounds, while the two novel enzymes carry out a similar reaction on biphenyl derivatives. The ring fission of 3,4-dihydroxybiphenyl by the purified enzymes results in the formation of (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid. These results suggest that the ring cleavage enzymes catalyzing phenol degradation are not involved in the ring cleavage of biarylic compounds by this yeast, although some intermediates of the phenol metabolism may function as inducers.

  4. Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

    PubMed Central

    Naing, Swe-Htet; Kalyoncu, Sibel; Smalley, David M.; Kim, Hyojung; Tao, Xingjian; George, Josh B.; Jonke, Alex P.; Oliver, Ryan C.; Urban, Volker S.; Torres, Matthew P.; Lieberman, Raquel L.

    2018-01-01

    Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His–Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr–Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aβ42 peptide (Ala–Thr) and the other to the non-pathogenic Aβ48 (Thr–Leu). For the former site, we observed more favorable kinetics in lipid bilayer–mimicking bicelles than in detergent solution, indicating that substrate–lipid and substrate–enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions. PMID:29382721

  5. Cell elongation is an adaptive response for clearing long chromatid arms from the cleavage plane

    PubMed Central

    Kotadia, Shaila; Montembault, Emilie; Sullivan, William

    2012-01-01

    Chromosome segregation must be coordinated with cell cleavage to ensure correct transmission of the genome to daughter cells. Here we identify a novel mechanism by which Drosophila melanogaster neuronal stem cells coordinate sister chromatid segregation with cleavage furrow ingression. Cells adapted to a dramatic increase in chromatid arm length by transiently elongating during anaphase/telophase. The degree of cell elongation correlated with the length of the trailing chromatid arms and was concomitant with a slight increase in spindle length and an enlargement of the zone of cortical myosin distribution. Rho guanine-nucleotide exchange factor (Pebble)–depleted cells failed to elongate during segregation of long chromatids. As a result, Pebble-depleted adult flies exhibited morphological defects likely caused by cell death during development. These studies reveal a novel pathway linking trailing chromatid arms and cortical myosin that ensures the clearance of chromatids from the cleavage plane at the appropriate time during cytokinesis, thus preserving genome integrity. PMID:23185030

  6. Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses.

    PubMed

    Davis, David A; Naiman, Nicole E; Wang, Victoria; Shrestha, Prabha; Haque, Muzammel; Hu, Duosha; Anagho, Holda A; Carey, Robert F; Davidoff, Katharine S; Yarchoan, Robert

    2015-07-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited in vitro and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1 beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome

  7. Regulation of adipolin/CTRP12 cleavage by obesity.

    PubMed

    Enomoto, Takashi; Shibata, Rei; Ohashi, Koji; Kambara, Takahiro; Kataoka, Yoshiyuki; Uemura, Yusuke; Yuasa, Daisuke; Murohara, Toyoaki; Ouchi, Noriyuki

    2012-11-09

    Obesity is highly associated with the development of insulin resistance and type 2 diabetes. Recently we found that adipolin/CRTP12 is an adipocytokine that exerts beneficial actions on glucose metabolism. Here we investigated the regulation of circulating adipolin under conditions of obesity and assessed its potential mechanisms. Both full and cleaved forms of adipolin were observed in mouse plasma. Diet-induced obese (DIO) mice showed a significant reduction of plasma levels of full and total (full and cleaved) adipolin compared with control mice, resulting in an increase in the ratio of cleaved to full isoform. In vitro gene transfection studies using HEK293 cells revealed that a deletion mutant of adipolin gene (Δaa90-93) caused a reduction of cleaved production of adipolin in media. A bioinformatics analysis of adipolin amino acid sequence indicated the potential involvement of the family of proprotein convertases (PCs) in cleavage of adipolin. Treatment of 3T3-L1 adipocytes with an inhibitor for PCs abolished the expression of cleaved adipolin form in the media. The expression of furin, the member of PCs, was increased in adipose tissue of DIO mice. Furin expression was also increased in cultured adipocytes by treatment with an inducer of inflammation. These data suggest that obesity states facilitate the cleavage of adipolin presumably through upregulation of furin in adipose tissue. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly

    PubMed Central

    Ait-Goughoulte, Malika; Hourioux, Christophe; Patient, Romuald; Trassard, Sylvie; Brand, Denys; Roingeard, Philippe

    2006-01-01

    SUMMARY Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. We used this model to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. We designed several mutants as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. Our data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly. PMID:16528035

  9. TMPRSS2 Independency for Haemagglutinin Cleavage In Vivo Differentiates Influenza B Virus from Influenza A Virus

    PubMed Central

    Sakai, Kouji; Ami, Yasushi; Nakajima, Noriko; Nakajima, Katsuhiro; Kitazawa, Minori; Anraku, Masaki; Takayama, Ikuyo; Sangsriratanakul, Natthanan; Komura, Miyuki; Sato, Yuko; Asanuma, Hideki; Takashita, Emi; Komase, Katsuhiro; Takehara, Kazuaki; Tashiro, Masato; Hasegawa, Hideki; Odagiri, Takato; Takeda, Makoto

    2016-01-01

    Influenza A and B viruses show clear differences in their host specificity and pandemic potential. Recent studies have revealed that the host protease TMPRSS2 plays an essential role for proteolytic activation of H1, H3, and H7 subtype strains of influenza A virus (IAV) in vivo. IAV possessing a monobasic cleavage site in the haemagglutinin (HA) protein replicates poorly in TMPRSS2 knockout mice owing to insufficient HA cleavage. In the present study, human isolates of influenza B virus (IBV) strains and a mouse-adapted IBV strain were analysed. The data showed that IBV successfully underwent HA cleavage in TMPRSS2 knockout mice, and that the mouse-adapted strain was fully pathogenic to these mice. The present data demonstrate a clear difference between IAV and IBV in their molecular mechanisms for spreading in vivo. PMID:27389476

  10. TMPRSS2 Independency for Haemagglutinin Cleavage In Vivo Differentiates Influenza B Virus from Influenza A Virus.

    PubMed

    Sakai, Kouji; Ami, Yasushi; Nakajima, Noriko; Nakajima, Katsuhiro; Kitazawa, Minori; Anraku, Masaki; Takayama, Ikuyo; Sangsriratanakul, Natthanan; Komura, Miyuki; Sato, Yuko; Asanuma, Hideki; Takashita, Emi; Komase, Katsuhiro; Takehara, Kazuaki; Tashiro, Masato; Hasegawa, Hideki; Odagiri, Takato; Takeda, Makoto

    2016-07-08

    Influenza A and B viruses show clear differences in their host specificity and pandemic potential. Recent studies have revealed that the host protease TMPRSS2 plays an essential role for proteolytic activation of H1, H3, and H7 subtype strains of influenza A virus (IAV) in vivo. IAV possessing a monobasic cleavage site in the haemagglutinin (HA) protein replicates poorly in TMPRSS2 knockout mice owing to insufficient HA cleavage. In the present study, human isolates of influenza B virus (IBV) strains and a mouse-adapted IBV strain were analysed. The data showed that IBV successfully underwent HA cleavage in TMPRSS2 knockout mice, and that the mouse-adapted strain was fully pathogenic to these mice. The present data demonstrate a clear difference between IAV and IBV in their molecular mechanisms for spreading in vivo.

  11. Base substitutions at scissile bond sites are sufficient to alter RNA-binding and cleavage activity of RNase III.

    PubMed

    Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok

    2011-02-01

    RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  12. Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

    PubMed Central

    Narula, Gagandeep; Tse-Dinh, Yuk-Ching

    2012-01-01

    Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the −4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the −4 and the −5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the −4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the −4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein–DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway. PMID:22833607

  13. Unconjugated Bilirubin Inhibits Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Protease

    PubMed Central

    Lu, Rui-Nan; Yang, Shangbin; Wu, Haifeng M.; Zheng, X. Long

    2015-01-01

    Summary Background Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood. Objectives The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13. Methods Fluorogenic, SELDI-TOF mass spectrometric assay, and Western blotting analyses were employed to address this question. Results Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of ~13 μM, ~70 μM, and ~17 μM, respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia is lower prior to than after treatment with bilirubin oxidase. Conclusions unconjugated bilirubin directly inhibits ADAMTS13’s ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has an adverse effect in vivo remains to be determined in our future study. PMID:25782102

  14. Contributions to Future Stratospheric Climate Change: An Idealized Chemistry-Climate Model Sensitivity Study

    NASA Technical Reports Server (NTRS)

    Hurwitz, M. M.; Braesicke, P.; Pyle, J. A.

    2010-01-01

    Within the framework of an idealized model sensitivity study, three of the main contributors to future stratospheric climate change are evaluated: increases in greenhouse gas concentrations, ozone recovery, and changing sea surface temperatures (SSTs). These three contributors are explored in combination and separately, to test the interactions between ozone and climate; the linearity of their contributions to stratospheric climate change is also assessed. In a simplified chemistry-climate model, stratospheric global mean temperature is most sensitive to CO2 doubling, followed by ozone depletion, then by increased SSTs. At polar latitudes, the Northern Hemisphere (NH) stratosphere is more sensitive to changes in CO2, SSTs and O3 than is the Southern Hemisphere (SH); the opposing responses to ozone depletion under low or high background CO2 concentrations, as seen with present-day SSTs, are much weaker and are not statistically significant under enhanced SSTs. Consistent with previous studies, the strength of the Brewer-Dobson circulation is found to increase in an idealized future climate; SSTs contribute most to this increase in the upper troposphere/lower stratosphere (UT/LS) region, while CO2 and ozone changes contribute most in the stratosphere and mesosphere.

  15. Constitutive α- and β-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines.

    PubMed

    Colombo, Alessio; Wang, Huanhuan; Kuhn, Peer-Hendrik; Page, Richard; Kremmer, Elisabeth; Dempsey, Peter J; Crawford, Howard C; Lichtenthaler, Stefan F

    2013-01-01

    Proteolytic cleavage of the amyloid precursor protein (APP) by the two proteases α- and β-secretases controls the generation of the amyloid β peptide (Aβ), a key player in Alzheimer's disease pathogenesis. The α-secretase ADAM10 and the β-secretase BACE1 have opposite effects on Aβ generation and are assumed to compete for APP as a substrate, such that their cleavages are inversely coupled. This concept was mainly demonstrated in studies using activation or overexpression of α- and β-secretases. Here, we report that this inverse coupling is not seen to the same extent upon inhibition of the endogenous proteases. Genetic and pharmacological inhibition of ADAM10 and BACE1 revealed that the endogenous, constitutive α-secretase cleavage of APP is largely uncoupled from β-secretase cleavage and Aβ generation in neuroglioma H4 cells and in neuronally differentiated SH-SY5Y cells. In contrast, inverse coupling was observed in primary cortical neurons. However, this coupling was not bidirectional. Inhibition of BACE1 increased ADAM10 cleavage of APP, but a reduction of ADAM10 activity did not increase the BACE1 cleavage of APP in the neurons. Our analysis shows that the inverse coupling of the endogenous α- and β-secretase cleavages depends on the cellular model and suggests that a reduction of ADAM10 activity is unlikely to increase the AD risk through increased β-secretase cleavage. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. [A time-lapse cinematographic analysis of ooplasmic movements during the cleavage ofPimpla turionellae L. (Hymenoptera)].

    PubMed

    Wolf, Rainer; Krause, Gerhard

    1971-09-01

    In the eggs ofPimpla turionellae, which are characterized by a long germ anlage ("long-germ egg" type), the cleavage nuclei primarily populate the anterior part and only later appear in the posterior of the egg lumen during the intravitelline cleavage. Gastrulation and segmentation also start within this anterior region. Time-lapse motion pictures served to observe and to check quantitatively even slow movements during cleavage and blastogenesis. In motion diagrams made by means of microkymographic technics the flow within the ooplasm along the longer axis of the egg has been timed.Shortly before the first cleavage in thestrictly unfertilized male eggs a short-time"unipolar flow" sets in from a primary initial region at 90% of their length. Thus a pillar of "central plasm" between both of the poles becomes shifted towards the posterior, while its outer coating layer of "marginal-plasm" is displaced forwards by the same distance. In eggs from fertilized females two successive flows of the same "unipolar" type have been observed.At the end of the third cleavage the energids, heretofore loosely grouped together, become distributed within the central plasm to form a "nuclear column". At the same time a fluently pulsatory "bipolar flow" sets in, within asecondary initial region at 80% of the egg length. Comparable to two mirror-image fountains, parts of the central plasm are carried towards the front pole and to the rear pole of the egg, respectively, while the marginal plasm, together with the oolemma, flows in opposite directions at times. With each pulsation the moving areas of the bipolar flow are shifted more and more towards the egg poles. The occurrence of bipolar flow pulsations, amounting to five, is correlated with the nuclear divisions in a still unknown way. In the rhythm of the bipolar flow, the energids become dispersed within the central plasm with a certain spatial lagging.After the bipolar flow has come to a halt, four further cleavages are indicated by

  17. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

    PubMed Central

    Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

    2015-01-01

    Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

  18. The Statistical Mechanics of Ideal MHD Turbulence

    NASA Technical Reports Server (NTRS)

    Shebalin, John V.

    2003-01-01

    Turbulence is a universal, nonlinear phenomenon found in all energetic fluid and plasma motion. In particular. understanding magneto hydrodynamic (MHD) turbulence and incorporating its effects in the computation and prediction of the flow of ionized gases in space, for example, are great challenges that must be met if such computations and predictions are to be meaningful. Although a general solution to the "problem of turbulence" does not exist in closed form, numerical integrations allow us to explore the phase space of solutions for both ideal and dissipative flows. For homogeneous, incompressible turbulence, Fourier methods are appropriate, and phase space is defined by the Fourier coefficients of the physical fields. In the case of ideal MHD flows, a fairly robust statistical mechanics has been developed, in which the symmetry and ergodic properties of phase space is understood. A discussion of these properties will illuminate our principal discovery: Coherent structure and randomness co-exist in ideal MHD turbulence. For dissipative flows, as opposed to ideal flows, progress beyond the dimensional analysis of Kolmogorov has been difficult. Here, some possible future directions that draw on the ideal results will also be discussed. Our conclusion will be that while ideal turbulence is now well understood, real turbulence still presents great challenges.

  19. Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

    PubMed

    Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T; Scheaffer, Suzanne M; Roswit, William T; Alevy, Yael G; Patel, Anand C; Heier, Richard F; Romero, Arthur G; Nichols, Colin G; Holtzman, Michael J; Brett, Tom J

    2012-12-07

    The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

  20. Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    PubMed Central

    Redpath, G. M. I.; Woolger, N.; Piper, A. K.; Lemckert, F. A.; Lek, A.; Greer, P. A.; North, K. N.; Cooper, S. T.

    2014-01-01

    Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlinC72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a–specific antibody recognizing cleaved mini-dysferlinC72 intensely labels the circumference of injury sites, supporting a key role for dysferlinExon40a isoforms in membrane repair and consistent with our evidence suggesting that the calpain-cleaved C-terminal module is the form specifically recruited to injury sites. Calpain cleavage of dysferlin is a ubiquitous response to membrane injury in multiple cell lineages and occurs independently of the membrane repair protein MG53. Our study links calpain and dysferlin in the calcium-activated vesicle fusion of membrane repair, placing calpains as upstream mediators of a membrane repair cascade that elicits cleaved dysferlin as an effector. Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain. Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module. PMID:25143396

  1. Predicting Film Genres with Implicit Ideals

    PubMed Central

    Olney, Andrew McGregor

    2013-01-01

    We present a new approach to defining film genre based on implicit ideals. When viewers rate the likability of a film, they indirectly express their ideal of what a film should be. Across six studies we investigate the category structure that emerges from likability ratings and the category structure that emerges from the features of film. We further compare these data-driven category structures with human annotated film genres. We conclude that film genres are structured more around ideals than around features of film. This finding lends experimental support to the notion that film genres are set of shifting, fuzzy, and highly contextualized psychological categories. PMID:23423823

  2. Predicting film genres with implicit ideals.

    PubMed

    Olney, Andrew McGregor

    2012-01-01

    We present a new approach to defining film genre based on implicit ideals. When viewers rate the likability of a film, they indirectly express their ideal of what a film should be. Across six studies we investigate the category structure that emerges from likability ratings and the category structure that emerges from the features of film. We further compare these data-driven category structures with human annotated film genres. We conclude that film genres are structured more around ideals than around features of film. This finding lends experimental support to the notion that film genres are set of shifting, fuzzy, and highly contextualized psychological categories.

  3. Copper-catalyzed transformation of ketones to amides via C(CO)-C(alkyl) bond cleavage directed by picolinamide.

    PubMed

    Ma, Haojie; Zhou, Xiaoqiang; Zhan, Zhenzhen; Wei, Daidong; Shi, Chong; Liu, Xingxing; Huang, Guosheng

    2017-09-13

    Copper catalyzed chemoselective cleavage of the C(CO)-C(alkyl) bond leading to C-N bond formation with chelation assistance of N-containing directing groups is described. Inexpensive Cu(ii)-acetate serves as a convenient catalyst for this transformation. This method highlights the emerging strategy to transform unactivated alkyl ketones into amides in organic synthesis and provides a new strategy for C-C bond cleavage.

  4. Idealized vs. Realistic Microstructures: An Atomistic Simulation Case Study on γ/γ' Microstructures.

    PubMed

    Prakash, Aruna; Bitzek, Erik

    2017-01-23

    Single-crystal Ni-base superalloys, consisting of a two-phase γ / γ ' microstructure, retain high strengths at elevated temperatures and are key materials for high temperature applications, like, e.g., turbine blades of aircraft engines. The lattice misfit between the γ and γ ' phases results in internal stresses, which significantly influence the deformation and creep behavior of the material. Large-scale atomistic simulations that are often used to enhance our understanding of the deformation mechanisms in such materials must accurately account for such misfit stresses. In this work, we compare the internal stresses in both idealized and experimentally-informed, i.e., more realistic, γ / γ ' microstructures. The idealized samples are generated by assuming, as is frequently done, a periodic arrangement of cube-shaped γ ' particles with planar γ / γ ' interfaces. The experimentally-informed samples are generated from two different sources to produce three different samples-the scanning electron microscopy micrograph-informed quasi-2D atomistic sample and atom probe tomography-informed stoichiometric and non-stoichiometric atomistic samples. Additionally, we compare the stress state of an idealized embedded cube microstructure with finite element simulations incorporating 3D periodic boundary conditions. Subsequently, we study the influence of the resulting stress state on the evolution of dislocation loops in the different samples. The results show that the stresses in the atomistic and finite element simulations are almost identical. Furthermore, quasi-2D boundary conditions lead to a significantly different stress state and, consequently, different evolution of the dislocation loop, when compared to samples with fully 3D boundary conditions.

  5. High-valent manganese–oxo valence tautomers and the influence of Lewis/Brönsted acids on C–H bond cleavage

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Baglia, Regina A.; Krest, Courtney M.; Yang, Tzuhsiung

    The addition of Lewis or Brönsted acids (LA = Zn(OTf) 2, B(C 6F 5) 3, HBAr F, TFA) to the high-valent manganese–oxo complex Mn V(O)(TBP 8Cz) results in the stabilization of a valence tautomer Mn IV(O-LA)(TBP 8Cz •+). The Zn II and B(C 6F 5) 3 complexes were characterized by manganese K-edge X-ray absorption spectroscopy (XAS). The position of the edge energies and the intensities of the pre-edge (1s to 3d) peaks confirm that the Mn ion is in the +4 oxidation state. Fitting of the extended X-ray absorption fine structure (EXAFS) region reveals 4 N/O ligands at Mn–N avemore » = 1.89 Å and a fifth N/O ligand at 1.61 Å, corresponding to the terminal oxo ligand. This Mn–O bond length is elongated compared to the Mn V(O) starting material (Mn–O = 1.55 Å). The reactivity of Mn IV(O-LA)(TBP 8Cz •+) toward C–H substrates was examined, and it was found that H • abstraction from C–H bonds occurs in a 1:1 stoichiometry, giving a Mn IV complex and the dehydrogenated organic product. The rates of C–H cleavage are accelerated for the Mn IV(O-LA)(TBP 8Cz •+) valence tautomer as compared to the MnV(O) valence tautomer when LA = Zn II, B(C 6F 5) 3, and HBArF, whereas for LA = TFA, the C–H cleavage rate is slightly slower than when compared to MnV(O). A large, nonclassical kinetic isotope effect of k H/ k D = 25–27 was observed for LA = B(C 6F 5) 3 and HBAr F, indicating that H-atom transfer (HAT) is the rate-limiting step in the C–H cleavage reaction and implicating a potential tunneling mechanism for HAT. Furthermore, the reactivity of Mn IV(O-LA)(TBP 8Cz •+) toward C–H bonds depends on the strength of the Lewis acid. The HAT reactivity is compared with the analogous corrole complex Mn IV(O–H)(tpfc •+) recently reported.« less

  6. High-valent manganese–oxo valence tautomers and the influence of Lewis/Brönsted acids on C–H bond cleavage

    DOE PAGES

    Baglia, Regina A.; Krest, Courtney M.; Yang, Tzuhsiung; ...

    2016-09-30

    The addition of Lewis or Brönsted acids (LA = Zn(OTf) 2, B(C 6F 5) 3, HBAr F, TFA) to the high-valent manganese–oxo complex Mn V(O)(TBP 8Cz) results in the stabilization of a valence tautomer Mn IV(O-LA)(TBP 8Cz •+). The Zn II and B(C 6F 5) 3 complexes were characterized by manganese K-edge X-ray absorption spectroscopy (XAS). The position of the edge energies and the intensities of the pre-edge (1s to 3d) peaks confirm that the Mn ion is in the +4 oxidation state. Fitting of the extended X-ray absorption fine structure (EXAFS) region reveals 4 N/O ligands at Mn–N avemore » = 1.89 Å and a fifth N/O ligand at 1.61 Å, corresponding to the terminal oxo ligand. This Mn–O bond length is elongated compared to the Mn V(O) starting material (Mn–O = 1.55 Å). The reactivity of Mn IV(O-LA)(TBP 8Cz •+) toward C–H substrates was examined, and it was found that H • abstraction from C–H bonds occurs in a 1:1 stoichiometry, giving a Mn IV complex and the dehydrogenated organic product. The rates of C–H cleavage are accelerated for the Mn IV(O-LA)(TBP 8Cz •+) valence tautomer as compared to the MnV(O) valence tautomer when LA = Zn II, B(C 6F 5) 3, and HBArF, whereas for LA = TFA, the C–H cleavage rate is slightly slower than when compared to MnV(O). A large, nonclassical kinetic isotope effect of k H/ k D = 25–27 was observed for LA = B(C 6F 5) 3 and HBAr F, indicating that H-atom transfer (HAT) is the rate-limiting step in the C–H cleavage reaction and implicating a potential tunneling mechanism for HAT. Furthermore, the reactivity of Mn IV(O-LA)(TBP 8Cz •+) toward C–H bonds depends on the strength of the Lewis acid. The HAT reactivity is compared with the analogous corrole complex Mn IV(O–H)(tpfc •+) recently reported.« less

  7. Ideal Theory in Semigroups Based on Intersectional Soft Sets

    PubMed Central

    Song, Seok Zun; Jun, Young Bae

    2014-01-01

    The notions of int-soft semigroups and int-soft left (resp., right) ideals are introduced, and several properties are investigated. Using these notions and the notion of inclusive set, characterizations of subsemigroups and left (resp., right) ideals are considered. Using the notion of int-soft products, characterizations of int-soft semigroups and int-soft left (resp., right) ideals are discussed. We prove that the soft intersection of int-soft left (resp., right) ideals (resp., int-soft semigroups) is also int-soft left (resp., right) ideals (resp., int-soft semigroups). The concept of int-soft quasi-ideals is also introduced, and characterization of a regular semigroup is discussed. PMID:25101310

  8. The effect of yield strength and ductility to fatigue damage

    NASA Technical Reports Server (NTRS)

    Yeh, H. Y.

    1973-01-01

    The cumulative damage of aluminium alloys with different yield strength and various ductility due to seismic loads was studied. The responses of an idealized beam with a centered mass at one end and fixed at the other end to El Centro's and Taft's earthquakes are computed by assuming that the alloys are perfectly elastoplastic materials and by using numerical technique. Consequently, the corresponding residual plastic strain can be obtained from the stress-strain relationship. The revised Palmgren-Miner cumulative damage theorem is utilized to calculate the fatigue damage. The numerical results show that in certain cases, the high ductility materials are more resistant to seismic loads than the high yield strength materials. The results also show that if a structure collapse during the earthquake, the collapse always occurs in the very early stage.

  9. Field-theoretical approach to a dense polymer with an ideal binary mixture of clustering centers.

    PubMed

    Fantoni, Riccardo; Müller-Nedebock, Kristian K

    2011-07-01

    We propose a field-theoretical approach to a polymer system immersed in an ideal mixture of clustering centers. The system contains several species of these clustering centers with different functionality, each of which connects a fixed number segments of the chain to each other. The field theory is solved using the saddle point approximation and evaluated for dense polymer melts using the random phase approximation. We find a short-ranged effective intersegment interaction with strength dependent on the average segment density and discuss the structure factor within this approximation. We also determine the fractions of linkers of the different functionalities.

  10. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation

    PubMed Central

    Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H.; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A. PMID:28052131

  11. Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

    PubMed Central

    Chand, Mahesh Kumar; Nirwan, Neha; Diffin, Fiona M.; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D.; Saikrishnan, Kayarat

    2015-01-01

    Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission. PMID:26389736

  12. Fast cleavage of phycocyanobilin from phycocyanin for use in food colouring.

    PubMed

    Roda-Serrat, Maria Cinta; Christensen, Knud Villy; El-Houri, Rime Bahij; Fretté, Xavier; Christensen, Lars Porskjær

    2018-02-01

    Phycocyanins from cyanobacteria are possible sources for new natural blue colourants. Their chromophore, phycocyanobilin (PCB), was cleaved from the apoprotein by solvolysis in alcohols and alcoholic aqueous solutions. In all cases two PCB isomers were obtained, while different solvent adducts were formed upon the use of different reagents. The reaction is believed to take place via two competing pathways, a concerted E2 elimination and a S N 2 nucleophilic substitution. Three cleavage methods were compared in terms of yield and purity: conventional reflux, sealed vessel heated in an oil bath, and microwave assisted reaction. The sealed vessel method is a new approach for fast cleavage of PCB from phycocyanin and gave at 120°C the same yield within 30min compared to 16h by the conventional reflux method (P<0.05). In addition the sealed vessel method resulted in improved purity compared to the other methods. Microwave irradiation increased product degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.

    Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant tomore » the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.« less

  14. Mechanistic Insights into Ring Cleavage and Contraction of Benzene over a Titanium Hydride Cluster.

    PubMed

    Kang, Xiaohui; Luo, Gen; Luo, Lun; Hu, Shaowei; Luo, Yi; Hou, Zhaomin

    2016-09-14

    Carbon-carbon bond cleavage of benzene by transition metals is of great fundamental interest and practical importance, as this transformation is involved in the production of fuels and other important chemicals in the industrial hydrocracking of naphtha on solid catalysts. Although this transformation is thought to rely on cooperation of multiple metal sites, molecular-level information on the reaction mechanism has remained scarce to date. Here, we report the DFT studies of the ring cleavage and contraction of benzene by a molecular trinuclear titanium hydride cluster. Our studies suggest that the reaction is initiated by benzene coordination, followed by H2 release, C6H6 hydrometalation, repeated C-C and C-H bond cleavage and formation to give a MeC5H4 unit, and insertion of a Ti atom into the MeC5H4 unit with release of H2 to give a metallacycle product. The C-C bond cleavage and ring contraction of toluene can also occur in a similar fashion, though some details are different due to the presence of the methyl substituent. Obviously, the facile release of H2 from the metal hydride cluster to provide electrons and to alter the charge population at the metal centers, in combination with the flexible metal-hydride connections and dynamic redox behavior of the trimetallic framework, has enabled this unusual transformation to occur. This work has not only provided unprecedented insights into the activation and transformation of benzene over a multimetallic framework but it may also offer help in the design of new molecular catalysts for the activation and transformation of inactive aromatics.

  15. Developmentally programmed DNA splicing in Paramecium reveals short-distance crosstalk between DNA cleavage sites

    PubMed Central

    Gratias, Ariane; Lepère, Gersende; Garnier, Olivier; Rosa, Sarah; Duharcourt, Sandra; Malinsky, Sophie; Meyer, Eric; Bétermier, Mireille

    2008-01-01

    Somatic genome assembly in the ciliate Paramecium involves the precise excision of thousands of short internal eliminated sequences (IESs) that are scattered throughout the germline genome and often interrupt open reading frames. Excision is initiated by double-strand breaks centered on the TA dinucleotides that are conserved at each IES boundary, but the factors that drive cleavage site recognition remain unknown. A degenerate consensus was identified previously at IES ends and genetic analyses confirmed the participation of their nucleotide sequence in efficient excision. Even for wild-type IESs, however, variant excision patterns (excised or nonexcised) may be inherited maternally through sexual events, in a homology-dependent manner. We show here that this maternal epigenetic control interferes with the targeting of DNA breaks at IES ends. Furthermore, we demonstrate that a mutation in the TA at one end of an IES impairs DNA cleavage not only at the mutant end but also at the wild-type end. We conclude that crosstalk between both ends takes place prior to their cleavage and propose that the ability of an IES to adopt an excision-prone conformation depends on the combination of its nucleotide sequence and of additional determinants. PMID:18420657

  16. Maintaining ideal body weight counseling sessions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brammer, S.H.

    The purpose of this program is to provide employees with the motivation, knowledge and skills necessary to maintain ideal body weight throughout life. The target audience for this program, which is conducted in an industrial setting, is the employee 40 years of age or younger who is at or near his/her ideal body weight.

  17. Surface functions during mitosis. III. Quantitative analysis of ligand- receptor movement into the cleavage furrow: diffusion vs. flow

    PubMed Central

    1982-01-01

    The surface distribution of concanavalin A (Con A) bound to cell membrane receptors varies dramatically as a function of mitotic phase. The lectin is distributed diffusely on cells labeled and observed between mid-prophase and early anaphase, whereas cells observed in late anaphase or telophase demonstrate a marked accumulation of Con A- receptor complexes over the developing cleavage furrow (Berlin, Oliver, and Walter. 1978. Cell. 15:327-341). In this report, we first use a system based on video intensification fluorescence microscopy to describe the simultaneous changes in cell shape and in lectin-receptor complex topography during progression of single cells through the mitotic cycle. The video analysis establishes that fluorescein succinyl Con A (F-S Con A)-receptor complex redistribution begins coincident with the first appearance of the cleavage furrow and is essentially complete within 2-3 min. This remarkable redistribution of surface fluorescence occurs during only a modest change in cell shape from a sphere to a belted cylinder. It reflects the translocation of complexes and not the accumulation of excess labeled membrane in the cleavage furrow: first, bound fluorescent cholera toxin which faithfully outlines the plasma membrane is not accumulated in the cleavage furrow, and, second, electron microscopy of peroxidase-Con A labeled cells undergoing cleavage shows that there is a high linear density of lectin within the furrow while Con A is virtually eliminated from the poles. The rate of surface movement of F-S Con A was quantitated by photon counting during a repetitive series of laser-excited fluorescence scans across dividing cells. Results were analyzed in terms of two alternative models of movement: a flow model in which complexes moved unidirectionally at constant velocity, and a diffusion model in which complexes could diffuse freely but were trapped at the cleavage furrow. According to these models, the observed rates of accumulation were

  18. SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

    PubMed

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-12-15

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Motoneurons secrete angiogenin to induce RNA cleavage in astroglia.

    PubMed

    Skorupa, Alexandra; King, Matthew A; Aparicio, Isabela M; Dussmann, Heiko; Coughlan, Karen; Breen, Bridget; Kieran, Dairin; Concannon, Caoimhin G; Marin, Philippe; Prehn, Jochen H M

    2012-04-11

    Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder affecting motoneurons. Mutations in angiogenin, encoding a member of the pancreatic RNase A superfamily, segregate with ALS. We previously demonstrated that angiogenin administration shows promise as a neuroprotective therapeutic in studies using transgenic ALS mice and primary motoneuron cultures. Its mechanism of action and target cells in the spinal cord, however, are largely unknown. Using mixed motoneuron cultures, motoneuron-like NSC34 cells, and primary astroglia cultures as model systems, we here demonstrate that angiogenin is a neuronally secreted factor that is endocytosed by astroglia and mediates neuroprotection in paracrine. We show that wild-type angiogenin acts unidirectionally to induce RNA cleavage in astroglia, while the ALS-associated K40I mutant is also secreted and endocytosed, but fails to induce RNA cleavage. Angiogenin uptake into astroglia requires heparan sulfate proteoglycans, and engages clathrin-mediated endocytosis. We show that this uptake mechanism exists for mouse and human angiogenin, and delivers a functional RNase output. Moreover, we identify syndecan 4 as the angiogenin receptor mediating the selective uptake of angiogenin into astroglia. Our data provide new insights into the paracrine activities of angiogenin in the nervous system, and further highlight the critical role of non-neuronal cells in the pathogenesis of ALS.

  20. The Ideal Man and Woman According to University Students

    ERIC Educational Resources Information Center

    Weinstein, Lawrence; Laverghetta, Antonio V.; Peterson, Scott A.

    2009-01-01

    The present study determined if the ideal man has changed over the years and who and what the ideal woman is. We asked students at Cameron University to rate the importance of character traits that define the ideal man and woman. Subjects also provided examples of famous people exemplifying the ideal, good, average, and inferior man and woman. We…

  1. Influence of cryopreservation on perinatal outcome after blastocyst- vs cleavage-stage embryo transfer: systematic review and meta-analysis.

    PubMed

    Alviggi, C; Conforti, A; Carbone, I F; Borrelli, R; de Placido, G; Guerriero, S

    2018-01-01

    To compare the perinatal outcomes of singleton pregnancies resulting from blastocyst- vs cleavage-stage embryo transfer and to assess whether they differ between fresh and frozen embryo transfer cycles. A systematic review of the literature was carried out using the Scopus, MEDLINE and ISI Web of Science databases with no time restriction. We included only peer-reviewed articles involving humans, in which perinatal outcomes of singleton pregnancies after blastocyst-stage embryo transfer were compared with those after cleavage-stage embryo transfer. Primary outcomes were preterm birth before 37 weeks and low birth weight (< 2500 g). Secondary outcomes were very preterm birth before 32 weeks, very low birth weight (< 1500 g), small-for-gestational-age (SGA), large-for-gestational-age (LGA), perinatal mortality and congenital anomaly. A meta-analysis was performed using a random-effects model. Three subgroups were evaluated: fresh only, frozen only and fresh plus frozen embryo transfer cycles. From a total of 3928 articles identified, 14 were selected for qualitative/quantitative analysis. Significantly higher incidences of preterm birth < 37 weeks (11 studies, n = 106 629 participants; risk ratio (RR), 1.15 (95% CI, 1.05 - 1.25); P = 0.002) and very preterm birth < 32 weeks (seven studies, n = 103 742; RR, 1.16 (95% CI, 1.02-1.31); P = 0.03) were observed after blastocyst- than after cleavage-stage embryo transfer in fresh cycles. However, the risk of preterm and very preterm birth was similar after blastocyst- and cleavage-stage transfers in frozen and fresh plus frozen cycles. Overall effect size analysis revealed fewer SGA deliveries after blastocyst- compared with cleavage-stage transfer in fresh cycles but a similar number in frozen cycles. Conversely, more LGA deliveries were observed after blastocyst- compared with cleavage-stage transfer in frozen cycles (two studies, n = 39 044; RR, 1.18 (95% CI, 1

  2. Sequence-selective DNA cleavage by a chimeric metallopeptide.

    PubMed

    Kovacic, Roger T; Welch, Joel T; Franklin, Sonya J

    2003-06-04

    A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as

  3. Molecular insight into bacterial cleavage of oceanic dimethylsulfoniopropionate into dimethyl sulfide

    PubMed Central

    Li, Chun-Yang; Wei, Tian-Di; Zhang, Sheng-Hui; Chen, Xiu-Lan; Gao, Xiang; Wang, Peng; Xie, Bin-Bin; Su, Hai-Nan; Qin, Qi-Long; Zhang, Xi-Ying; Yu, Juan; Zhang, Hong-Hai; Zhou, Bai-Cheng; Yang, Gui-Peng; Zhang, Yu-Zhong

    2014-01-01

    The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI_1157. The structures of DddQ bound to an inhibitory molecule 2-(N-morpholino)ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a β-barrel fold structure and contains a Zn2+ ion and six highly conserved hydrophilic residues (Tyr120, His123, His125, Glu129, Tyr131, and His163) in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the β-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between “open” and “closed” states, serving as a gate for DMSP entry. We also propose a molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally important biogeochemical reaction. PMID:24395783

  4. Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis.

    PubMed

    Rousseau, Beth A; Hou, Zhonggang; Gramelspacher, Max J; Zhang, Yan

    2018-03-01

    The microbial CRISPR systems enable adaptive defense against mobile elements and also provide formidable tools for genome engineering. The Cas9 proteins are type II CRISPR-associated, RNA-guided DNA endonucleases that identify double-stranded DNA targets by sequence complementarity and protospacer adjacent motif (PAM) recognition. Here we report that the type II-C CRISPR-Cas9 from Neisseria meningitidis (Nme) is capable of programmable, RNA-guided, site-specific cleavage and recognition of single-stranded RNA targets and that this ribonuclease activity is independent of the PAM sequence. We define the mechanistic feature and specificity constraint for RNA cleavage by NmeCas9 and also show that nuclease null dNmeCas9 binds to RNA target complementary to CRISPR RNA. Finally, we demonstrate that NmeCas9-catalyzed RNA cleavage can be blocked by three families of type II-C anti-CRISPR proteins. These results fundamentally expand the targeting capacities of CRISPR-Cas9 and highlight the potential utility of NmeCas9 as a single platform to target both RNA and DNA. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. The developmental effects of media-ideal internalization and self-objectification processes on adolescents' negative body-feelings, dietary restraint, and binge eating.

    PubMed

    Dakanalis, Antonios; Carrà, Giuseppe; Calogero, Rachel; Fida, Roberta; Clerici, Massimo; Zanetti, Maria Assunta; Riva, Giuseppe

    2015-08-01

    Despite accumulated experimental evidence of the negative effects of exposure to media-idealized images, the degree to which body image, and eating related disturbances are caused by media portrayals of gendered beauty ideals remains controversial. On the basis of the most up-to-date meta-analysis of experimental studies indicating that media-idealized images have the most harmful and substantial impact on vulnerable individuals regardless of gender (i.e., "internalizers" and "self-objectifiers"), the current longitudinal study examined the direct and mediated links posited in objectification theory among media-ideal internalization, self-objectification, shame and anxiety surrounding the body and appearance, dietary restraint, and binge eating. Data collected from 685 adolescents aged between 14 and 15 at baseline (47 % males), who were interviewed and completed standardized measures annually over a 3-year period, were analyzed using a structural equation modeling approach. Results indicated that media-ideal internalization predicted later thinking and scrutinizing of one's body from an external observer's standpoint (or self-objectification), which then predicted later negative emotional experiences related to one's body and appearance. In turn, these negative emotional experiences predicted subsequent dietary restraint and binge eating, and each of these core features of eating disorders influenced each other. Differences in the strength of these associations across gender were not observed, and all indirect effects were significant. The study provides valuable information about how the cultural values embodied by gendered beauty ideals negatively influence adolescents' feelings, thoughts and behaviors regarding their own body, and on the complex processes involved in disordered eating. Practical implications are discussed.

  6. Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke

    Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to constructmore » an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.« less

  7. Anisotropic toughness and strength in graphene and its atomistic origin

    NASA Astrophysics Data System (ADS)

    Hossain, M. Zubaer; Ahmed, Tousif; Silverman, Benjamin; Khawaja, M. Shehroz; Calderon, Justice; Rutten, Andrew; Tse, Stanley

    2018-01-01

    This paper presents the implication of crystallographic orientation on toughness and ideal strength in graphene under lattice symmetry-preserving and symmetry-breaking deformations. In symmetry-preserving deformation, both toughness and strength are isotropic, regardless of the chirality of the lattice; whereas, in symmetry-breaking deformation they are strongly anisotropic, even in the presence of vacancy defects. The maximum and minimum of toughness or strength occur for loading along the zigzag direction and the armchair direction, respectively. The anisotropic behavior is governed by a complex interplay among bond-stretching deformation, bond-bending deformation, and the chirality of the lattice. Nevertheless, the condition for crack-nucleation is dictated by the maximum bond-force required for bond rupture, and it is independent of the chiral angle of the lattice or loading direction. At the onset of crack-nucleation a localized nucleation zone is formed, wherein the bonds rupture locally satisfying the maximum bond-force criterion. The nucleation zone acts as the physical origin in triggering the fracture nucleation process, but its presence is undetectable from the macroscopic stress-strain data.

  8. Cleavage/alteration of interleukin-8 by matrix metalloproteinase-9 in the female lower genital tract.

    PubMed

    Zariffard, M Reza; Anastos, Kathryn; French, Audrey L; Munyazesa, Elisaphane; Cohen, Mardge; Landay, Alan L; Spear, Gregory T

    2015-01-01

    Interleukin-8 (IL-8, CXCL8) plays important roles in immune responses at mucosal sites including in the lower genital tract. Since several types of bacteria produce proteases that cleave IL-8 and many types of bacteria can be present in lower genital tract microbiota, we assessed genital fluids for IL-8 cleavage/alteration. Genital fluids collected by lavage from 200 women (23 HIV-seronegative and 177 HIV-seropositive) were tested for IL-8 cleavage/alteration by ELISA. IL-8 cleaving/altering activity was observed in fluids from both HIV-positive (28%) and HIV-negative women (35%). There was no clear relationship between the activity and the types of bacteria present in the lower genital tract as determined by high-throughput sequencing of the 16S rRNA gene. Protease inhibitors specific for matrix metalloproteinases (MMPs) reduced the activity and a multiplex assay that detects both inactive and active MMPs showed the presence of multiple MMPs, including MMP-1, -3, -7, -8, -9, -10 and -12 in genital secretions from many of the women. The IL-8-cleaving/altering activity significantly correlated with active MMP-9 as well as with cleavage of a substrate that is acted on by several active MMPs. These studies show that multiple MMPs are present in the genital tract of women and strongly suggest that MMP-9 in genital secretions can cleave IL-8 at this mucosal site. These studies suggest that MMP-mediated cleavage of IL-8 can modulate inflammatory responses in the lower genital tract.

  9. Identification of Proteolytic Cleavage Sites of EphA2 by Membrane Type 1 Matrix Metalloproteinase on the Surface of Cancer Cells.

    PubMed

    Kikuchi, Keiji; Kozuka-Hata, Hiroko; Oyama, Masaaki; Seiki, Motoharu; Koshikawa, Naohiko

    2018-01-01

    Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification . The purified fragment was digested with trypsin to generate proteolytic peptides , and the amino acid sequences of these peptides were determined by nano-LC-mass spectrometry to identify the MT1-MMP-mediated cleavage site(s) of EphA2.

  10. Neural transcription factors bias cleavage stage blastomeres to give rise to neural ectoderm

    PubMed Central

    Gaur, Shailly; Mandelbaum, Max; Herold, Mona; Majumdar, Himani Datta; Neilson, Karen M.; Maynard, Thomas M.; Mood, Kathy; Daar, Ira O.; Moody, Sally A.

    2016-01-01

    The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn and Sox11 each induced explants made from ventral, epidermis-producing blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activity is required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing blastomeres significantly increased expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage blastomeres to a neural fate. We demonstrate that mouse and human homologues of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and blastomere explants. PMID:27092474

  11. Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation*♦

    PubMed Central

    Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T.; Scheaffer, Suzanne M.; Roswit, William T.; Alevy, Yael G.; Patel, Anand C.; Heier, Richard F.; Romero, Arthur G.; Nichols, Colin G.; Holtzman, Michael J.; Brett, Tom J.

    2012-01-01

    The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface. PMID:23112050

  12. A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

    PubMed Central

    Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

  13. Cryotop vitrification as compared to conventional slow freezing for human embryos at the cleavage stage: survival and outcomes.

    PubMed

    Lin, Tseng-Kai; Su, Jin-Tsung; Lee, Fa-Kung; Lin, Yu-Ru; Lo, Hsiao-Ching

    2010-09-01

    This study was conducted to compare the efficacy of cryotop vitrification of human cleavage-stage embryos to that of conventional slow freezing of these embryos with respect to survival. A second objective was to compare the two cryopreservation techniques with respect to outcomes for a cohort of women. Cleavage-stage embryos from 102 patients were cryopreserved either by vitrification (57 patients) or by traditional slow freezing (45 patients). After thawing, rates of embryo survival, implantation, and clinical pregnancy were determined. Survival of embryos was significantly higher with the vitrification procedure as compared to traditional slow freezing [287/298 (96.3%) vs. 294/446 (65.9%); p < 0.05). Rates of implantation and clinical pregnancy were also significantly higher using vitrification procedure as compared to the slow freezing procedure (24.3% vs. 7.1% and 35.6% vs. 15.6% respectively, p < 0.05). As compared to conventional slow freezing, cryopreservation of human cleavage-stage embryo using vitrification results in higher rates of embryo survival, implantation, and clinical pregnancy. Vitrification therefore represents the superior cryopreservation technique for cleavage-stage embryos. Copyright © 2010 Taiwan Association of Obstetric & Gynecology. Published by Elsevier B.V. All rights reserved.

  14. Crenulation cleavage development by partitioning of deformation into zones of progressive shearing (combined shearing, shortening and volume loss) and progressive shortening (no volume loss): quantification of solution shortening and intermicrolithon-movement

    NASA Astrophysics Data System (ADS)

    Stewart, L. K.

    1997-11-01

    An analytical method for determining amounts of cleavage-normal dissolution and cleavage-parallel shear movement that occurred between adjacent microlithons during crenulation cleavage seam formation within a deformed slate is developed for the progressive bulk inhomogeneous shortening (PBIS) mechanism of crenulation cleavage formation. The method utilises structural information obtained from samples where a diverging bed and vein are offset by a crenulation cleavage seam. Several samples analysed using this method produced ratios of relative, cleavage-parallel movement of microlithons to the material thickness removed by dissolution typically in the range of 1.1-3.4:1. The mean amount of solution shortening attributed to the formation of the cleavage seams examined is 24%. The results indicate that a relationship may exist between the width of microlithons and the amount of cleavage-parallel intermicrolithon-movement. The method presented here has the potential to help determine whether crenulation cleavage seams formed by the progressive bulk inhomogeneous shortening mechanism or by that involving cleavage-normal pressure solution alone.

  15. Functional characterisation of three members of the Vitis vinifera L. carotenoid cleavage dioxygenase gene family

    PubMed Central

    2013-01-01

    Background In plants, carotenoids serve as the precursors to C13-norisoprenoids, a group of apocarotenoid compounds with diverse biological functions. Enzymatic cleavage of carotenoids catalysed by members of the carotenoid cleavage dioxygenase (CCD) family has been shown to produce a number of industrially important volatile flavour and aroma apocarotenoids including β-ionone, geranylacetone, pseudoionone, α-ionone and 3-hydroxy-β-ionone in a range of plant species. Apocarotenoids contribute to the floral and fruity attributes of many wine cultivars and are thereby, at least partly, responsible for the “varietal character”. Despite their importance in grapes and wine; carotenoid cleavage activity has only been described for VvCCD1 and the mechanism(s) and regulation of carotenoid catabolism remains largely unknown. Results Three grapevine-derived CCD-encoding genes have been isolated and shown to be functional with unique substrate cleavage capacities. Our results demonstrate that the VvCCD4a and VvCCD4b catalyse the cleavage of both linear and cyclic carotenoid substrates. The expression of VvCCD1, VvCCD4a and VvCCD4b was detected in leaf, flower and throughout berry development. VvCCD1 expression was constitutive, whereas VvCCD4a expression was predominant in leaves and VvCCD4b in berries. A transgenic population with a 12-fold range of VvCCD1 expression exhibited a lack of correlation between VvCCD1 expression and carotenoid substrates and/or apocarotenoid products in leaves, providing proof that the in planta function(s) of VvCCD1 in photosynthetically active tissue is distinct from the in vitro activities demonstrated. The isolation and functional characterisation of VvCCD4a and VvCCD4b identify two additional CCDs that are functional in grapevine. Conclusions Taken together, our results indicate that the three CCDs are under various levels of control that include gene expression (spatial and temporal), substrate specificity and compartmentalisation

  16. Dendritic polymer imaging systems for the evaluation of conjugate uptake and cleavage

    NASA Astrophysics Data System (ADS)

    Krüger, Harald R.; Nagel, Gregor; Wedepohl, Stefanie; Calderón, Marcelo

    2015-02-01

    Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening.Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular

  17. Idealism and materialism in perception.

    PubMed

    Rose, David; Brown, Dora

    2015-01-01

    Koenderink (2014, Perception, 43, 1-6) has said most Perception readers are deluded, because they believe an 'All Seeing Eye' observes an objective reality. We trace the source of Koenderink's assertion to his metaphysical idealism, and point to two major weaknesses in his position-namely, its dualism and foundationalism. We counter with arguments from modern philosophy of science for the existence of an objective material reality, contrast Koenderink's enactivism to his idealism, and point to ways in which phenomenology and cognitive science are complementary and not mutually exclusive.

  18. A novel quantitative electrochemical method to monitor DNA double-strand breaks caused by a DNA cleavage agent at a DNA sensor.

    PubMed

    Banasiak, Anna; Cassidy, John; Colleran, John

    2018-06-01

    To date, DNA cleavage, caused by cleavage agents, has been monitored mainly by gel and capillary electrophoresis. However, these techniques are time-consuming, non-quantitative and require gel stains. In this work, a novel, simple and, importantly, a quantitative method for monitoring the DNA nuclease activity of potential anti-cancer drugs, at a DNA electrochemical sensor, is presented. The DNA sensors were prepared using thiol-modified oligonucleotides that self-assembled to create a DNA monolayer at gold electrode surfaces. The quantification of DNA double-strand breaks is based on calculating the DNA surface coverage, before and after exposure to a DNA cleavage agent. The nuclease properties of a model DNA cleavage agent, copper bis-phenanthroline ([Cu II (phen) 2 ] 2+ ), that can cleave DNA in a Fenton-type reaction, were quantified electrochemically. The DNA surface coverage decreased on average by 21% after subjecting the DNA sensor to a nuclease assay containing [Cu II (phen) 2 ] 2+ , a reductant and an oxidant. This percentage indicates that 6 base pairs were cleaved in the nuclease assay from the immobilised 30 base pair strands. The DNA cleavage can be also induced electrochemically in the absence of a chemical reductant. [Cu II (phen) 2 ] 2+ intercalates between DNA base pairs and, on application of a suitable potential, can be reduced to [Cu I (phen) 2 ] + , with dissolved oxygen acting as the required oxidant. This reduction process is facilitated through DNA strands via long-range electron transfer, resulting in DNA cleavage of 23%. The control measurements for both chemically and electrochemically induced cleavage revealed that DNA strand breaks did not occur under experimental conditions in the absence of [Cu II (phen) 2 ] 2+ . Copyright © 2018 Elsevier B.V. All rights reserved.

  19. The role of GyrB in the DNA cleavage-religation reaction of DNA gyrase: a proposed two metal-ion mechanism.

    PubMed

    Noble, Christian G; Maxwell, Anthony

    2002-04-26

    We have examined the role of the DNA gyrase B protein in cleavage and religation of DNA using site-directed mutagenesis. Three aspartate residues and a glutamate residue: E424, D498, D500 and D502, thought to co-ordinate a magnesium ion, were mutated to alanine; in addition, the glutamate residue and one aspartate residue were mutated to glutamine and asparagine, respectively. We have shown that these residues are important for the cleavage-religation reaction and are likely to be involved in magnesium ion co-ordination. On separate mutation of two of these aspartate residues to cysteine or histidine, the metal ion preference for the DNA relaxation activity of gyrase changed from magnesium to manganese (II). We present evidence to support the idea that cleavage of each DNA strand involves two or more metal ions, and suggest a scheme for the DNA cleavage chemistry of DNA gyrase involving two metal ions. (c) 2002 Elsevier Science Ltd.

  20. The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors.

    PubMed

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-11-15

    Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

    PubMed Central

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-01-01

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

  2. Idealized vs. Realistic Microstructures: An Atomistic Simulation Case Study on γ/γ′ Microstructures

    PubMed Central

    Prakash, Aruna; Bitzek, Erik

    2017-01-01

    Single-crystal Ni-base superalloys, consisting of a two-phase γ/γ′ microstructure, retain high strengths at elevated temperatures and are key materials for high temperature applications, like, e.g., turbine blades of aircraft engines. The lattice misfit between the γ and γ′ phases results in internal stresses, which significantly influence the deformation and creep behavior of the material. Large-scale atomistic simulations that are often used to enhance our understanding of the deformation mechanisms in such materials must accurately account for such misfit stresses. In this work, we compare the internal stresses in both idealized and experimentally-informed, i.e., more realistic, γ/γ′ microstructures. The idealized samples are generated by assuming, as is frequently done, a periodic arrangement of cube-shaped γ′ particles with planar γ/γ′ interfaces. The experimentally-informed samples are generated from two different sources to produce three different samples—the scanning electron microscopy micrograph-informed quasi-2D atomistic sample and atom probe tomography-informed stoichiometric and non-stoichiometric atomistic samples. Additionally, we compare the stress state of an idealized embedded cube microstructure with finite element simulations incorporating 3D periodic boundary conditions. Subsequently, we study the influence of the resulting stress state on the evolution of dislocation loops in the different samples. The results show that the stresses in the atomistic and finite element simulations are almost identical. Furthermore, quasi-2D boundary conditions lead to a significantly different stress state and, consequently, different evolution of the dislocation loop, when compared to samples with fully 3D boundary conditions. PMID:28772453

  3. Medical learning curves and the Kantian ideal.

    PubMed

    Le Morvan, P; Stock, B

    2005-09-01

    A hitherto unexamined problem for the "Kantian ideal" that one should always treat patients as ends in themselves, and never only as a means to other ends, is explored in this paper. The problem consists of a prima facie conflict between this Kantian ideal and the reality of medical practice. This conflict arises because, at least presently, medical practitioners can only acquire certain skills and abilities by practising on live, human patients, and given the inevitability and ubiquity of learning curves, this learning requires some patients to be treated only as a means to this end. A number of ways of attempting to establish the compatibility of the Kantian Ideal with the reality of medical practice are considered. Each attempt is found to be unsuccessful. Accordingly, until a way is found to reconcile them, we conclude that the Kantian ideal is inconsistent with the reality of medical practice.

  4. Medical learning curves and the Kantian ideal

    PubMed Central

    Le Morvan, P; Stock, B

    2005-01-01

    A hitherto unexamined problem for the "Kantian ideal" that one should always treat patients as ends in themselves, and never only as a means to other ends, is explored in this paper. The problem consists of a prima facie conflict between this Kantian ideal and the reality of medical practice. This conflict arises because, at least presently, medical practitioners can only acquire certain skills and abilities by practising on live, human patients, and given the inevitability and ubiquity of learning curves, this learning requires some patients to be treated only as a means to this end. A number of ways of attempting to establish the compatibility of the Kantian Ideal with the reality of medical practice are considered. Each attempt is found to be unsuccessful. Accordingly, until a way is found to reconcile them, we conclude that the Kantian ideal is inconsistent with the reality of medical practice. PMID:16131552

  5. The caspase-generated cleavage product of Ets-1 p51 and Ets-1 p27, Cp17, induces apoptosis.

    PubMed

    Choul-Li, Souhaila; Tulasne, David; Aumercier, Marc

    2016-11-04

    The transcription factor Ets-1 is involved in various physiological processes and invasive pathologies. Human Ets-1 exists under three isoforms: p51, the predominant full-length isoform, p42 and p27, shorter alternatively spliced isoforms. We have previously demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed by caspases in vitro and during apoptosis. However, the caspase cleavage of the second spliced variant Ets-1 p27 remains to investigate. In the present study, we demonstrate that Ets-1 p27 is a cleavage substrate of caspases. We show that Ets-1 p27 is processed in vitro by caspase-3, resulting in three C-terminal fragments Cp20, Cp17 and Cp14. Similarly, Ets-1 p27 was cleaved during apoptotic cell death induced by anisomycin, producing fragments consistent with those observed in in vitro cleavage assay. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p27. As a functional consequences, Cp17 fragment, the major cleavage product generated during apoptosis, induced itself apoptosis when transfected into cells. Our results show that Ets-1 p27 is cleaved in the same manner as Ets-1 p51 within the exon VII-encoded region, thus generating a stable C-terminal fragment that induces cell death by initiating apoptosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Improving the prospects of cleavage-based nanopore sequencing engines

    NASA Astrophysics Data System (ADS)

    Brady, Kyle T.; Reiner, Joseph E.

    2015-08-01

    Recently proposed methods for DNA sequencing involve the use of cleavage-based enzymes attached to the opening of a nanopore. The idea is that DNA interacting with either an exonuclease or polymerase protein will lead to a small molecule being cleaved near the mouth of the nanopore, and subsequent entry into the pore will yield information about the DNA sequence. The prospects for this approach seem promising, but it has been shown that diffusion related effects impose a limit on the capture probability of molecules by the pore, which limits the efficacy of the technique. Here, we revisit the problem with the goal of optimizing the capture probability via a step decrease in the nucleotide diffusion coefficient between the pore and bulk solutions. It is shown through random walk simulations and a simplified analytical model that decreasing the molecule's diffusion coefficient in the bulk relative to its value in the pore increases the nucleotide capture probability. Specifically, we show that at sufficiently high applied transmembrane potentials (≥100 mV), increasing the potential by a factor f is equivalent to decreasing the diffusion coefficient ratio Dbulk/Dpore by the same factor f. This suggests a promising route toward implementation of cleavage-based sequencing protocols. We also discuss the feasibility of forming a step function in the diffusion coefficient across the pore-bulk interface.

  7. Formation of Pmel17 Amyloid Is Regulated by Juxtamembrane Metalloproteinase Cleavage, and the Resulting C-terminal Fragment Is a Substrate for γ-Secretase*

    PubMed Central

    Kummer, Markus P.; Maruyama, Hiroko; Huelsmann, Claudia; Baches, Sandra; Weggen, Sascha; Koo, Edward H.

    2009-01-01

    The formation of insoluble cross β-sheet amyloid is pathologically associated with disorders such as Alzheimer, Parkinson, and Huntington diseases. One exception is the nonpathological amyloid derived from the protein Pmel17 within melanosomes to generate melanin pigment. Here we show that the formation of insoluble MαC intracellular fragments of Pmel17, which are the direct precursors to Pmel17 amyloid, depends on a novel juxtamembrane cleavage at amino acid position 583 between the furin-like proprotein convertase cleavage site and the transmembrane domain. The resulting Pmel17 C-terminal fragment is then processed by the γ-secretase complex to release a short-lived intracellular domain fragment. Thus, by analogy to the Notch receptor, we designate this cleavage the S2 cleavage site, whereas γ-secretase mediates proteolysis at the intramembrane S3 site. Substitutions or deletions at this S2 cleavage site, the use of the metalloproteinase inhibitor TAPI-2, as well as small interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17 reduced the formation of insoluble Pmel17 fragments. These results demonstrate that the release of the Pmel17 ectodomain, which is critical for melanin amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position. PMID:19047044

  8. Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase.

    PubMed

    Chan, Pan F; Germe, Thomas; Bax, Benjamin D; Huang, Jianzhong; Thalji, Reema K; Bacqué, Eric; Checchia, Anna; Chen, Dongzhao; Cui, Haifeng; Ding, Xiao; Ingraham, Karen; McCloskey, Lynn; Raha, Kaushik; Srikannathasan, Velupillai; Maxwell, Anthony; Stavenger, Robert A

    2017-05-30

    A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.

  9. Power of Orbitrap-based LC-high resolution-MS/MS for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on-spot cleavage in comparison to established LC-MSn or GC-MS procedures.

    PubMed

    Michely, Julian A; Meyer, Markus R; Maurer, Hans H

    2018-01-01

    Reliable, sensitive, and comprehensive urine screening procedures by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) with low or high resolution (HR) are of high importance for drug testing, adherence monitoring, or detection of toxic compounds. Besides conventional urine sampling, dried urine spots are of increasing interest. In the present study, the power of LC-HR-MS/MS was investigated for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on-spot cleavage in comparison to established LC-MS n or GC-MS procedures. Authentic human urine samples (n = 103) were split in 4 parts. One aliquot was prepared by precipitation (UP), one by UP with conjugate cleavage (UglucP), one spot on filter paper cards and prepared by on-spot cleavage followed by liquid extraction (DUSglucE), and one worked-up by acid hydrolysis, liquid-liquid extraction, and acetylation for GC-MS analysis. The 3 series of LC-HR-MS/MS results were compared among themselves, to corresponding published LC-MS n data, and to screening results obtained by conventional GC-MS. The reference libraries used for the 3 techniques contained over 4500 spectra of parent compounds and their metabolites. The number of all detected hits (770 drug intakes) was set to 100%. The LC-HR-MS/MS approach detected 80% of the hits after UP, 89% after UglucP, and 77% after DUSglucE, which meant over one-third more hits in comparison to the corresponding published LC-MS n results with ≤49% detected hits. The GC-MS approach identified 56% of all detected hits. In conclusion, LC-HR-MS/MS provided the best screening results after conjugate cleavage and precipitation. Copyright © 2017 John Wiley & Sons, Ltd.

  10. [The style of leadership idealized by nurses].

    PubMed

    Higa, Elza de Fátima Ribeiro; Trevizan, Maria Auxiliadora

    2005-01-01

    This study focuses on nursing leadership on the basis of Grid theories. According to the authors, these theories are an alternative that allows for leadership development in nursing. The research aimed to identify and analyze the style of leadership idealized by nurses, according to their own view, and to compare the styles of leadership idealized by nurses between the two research institutions. Study subjects were 13 nurses. The results show that nurses at both institutions equally mention they idealize style 9.9, followed by 5.5 and 1.9, with a tendency to reject styles 9.1 and 1.1.

  11. Rubber Oxygenase and Latex Clearing Protein Cleave Rubber to Different Products and Use Different Cleavage Mechanisms

    PubMed Central

    Birke, Jakob

    2014-01-01

    Two types of enzyme for oxidative cleavage of poly(cis-1,4-isoprene) are known. One is rubber oxygenase (RoxA) that is secreted by Xanthomonas sp. strain 35Y and a few other Gram-negative rubber-degrading bacteria during growth on polyisoprene. RoxA was studied in the past, and the recently solved structure showed a structural relationship to bacterial cytochrome c peroxidases (J. Seidel et al., Proc. Natl. Acad. Sci. U. S. A. 110:13833–13838, 2013, http://dx.doi.org/10.1073/pnas.1305560110). The other enzyme is latex-clearing protein (Lcp) that is secreted by rubber-degrading actinomycetes, but Lcp has not yet been purified. Here, we expressed Lcp of Streptomyces sp. strain K30 in a ΔroxA background of Xanthomonas sp. strain 35Y and purified native (untagged) Lcp. The specific activities of Lcp and RoxA were 0.70 and 0.48 U/mg, respectively. Lcp differed from RoxA in the absence of heme groups and other characteristics. Notably, Lcp degraded polyisoprene via endo-type cleavage to tetra-C20 and higher oligo-isoprenoids with aldehyde and keto end groups, whereas RoxA used an exo-type cleavage mechanism to give the main end product 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). RoxA was able to cleave isolated Lcp-derived oligo-isoprenoid molecules to ODTD. Inhibitor studies, spectroscopic investigations and metal analysis gave no indication for the presence of iron, other metals, or cofactors in Lcp. Our results suggest that Lcp could be a member of the growing group of cofactor-independent oxygenases and differs in the cleavage mechanism from heme-dependent RoxA. In conclusion, RoxA and Lcp represent two different answers to the same biochemical problem, the cleavage of polyisoprene, a polymer that has carbon-carbon double bonds as the only functional groups for enzymatic attack. PMID:24907333

  12. Dramatic Improvement of the Mechanical Strength of Silane-Modified Hydroxyapatite–Gelatin Composites via Processing with Cosolvent

    PubMed Central

    2018-01-01

    Bone tissue engineering (BTE) requires a sturdy biomaterial for scaffolds for restoration of large bone defects. Ideally, the scaffold should have a mechanical strength comparable to the natural bone in the implanted site. We show that adding cosolvent during the processing of our previously developed composite of hydroxyapatite–gelatin with a silane cross-linker can significantly affect its mechanical strength. When processed with tetrahydrofuran (THF) as the cosolvent, the new hydroxyapatite–gelatin composite can demonstrate almost twice the compressive strength (97 vs 195 MPa) and biaxial flexural strength (222 vs 431 MPa) of the previously developed hydroxyapatite–gelatin composite (i.e., processed without THF), respectively. We further confirm that this mechanical strength improvement is due to the improved morphology of both the enTMOS network and the composite. Furthermore, the addition of cosolvents does not appear to negatively impact the cell viability. Finally, the porous scaffold can be easily fabricated, and its compressive strength is around 11 MPa under dry conditions. All these results indicate that this new hydroxyapatite–gelatin composite is a promising material for BTE application. PMID:29623305

  13. Ferromagnetic nanoparticles with peroxidase-like activity enhance the cleavage of biological macromolecules for biofilm elimination

    NASA Astrophysics Data System (ADS)

    GaoCurrent Address: University Of Pennsylvania, School Of Dental Medicine, Philadelphia, Pa 19104, Usa. E.-Mail: Gaoliz@Dental. Upenn. Edu, Lizeng; Giglio, Krista M.; Nelson, Jacquelyn L.; Sondermann, Holger; Travis, Alexander J.

    2014-02-01

    Hydrogen peroxide (H2O2) is a ``green chemical'' that has various cleaning and disinfectant uses, including as an anti-bacterial agent for hygienic and medical treatments. However, its efficacy is limited against biofilm-producing bacteria, because of poor penetration into the protective, organic matrix. Here we show new applications for ferromagnetic nanoparticles (Fe3O4, MNPs) with peroxidase-like activity in potentiating the efficacy of H2O2 in biofilm degradation and prevention. Our data show that MNPs enhanced oxidative cleavage of biofilm components (model nucleic acids, proteins, and oligosaccharides) in the presence of H2O2. When challenged with live, biofilm-producing bacteria, the MNP-H2O2 system efficiently broke down the existing biofilm and prevented new biofilms from forming, killing both planktonic bacteria and those within the biofilm. By enhancing oxidative cleavage of various substrates, the MNP-H2O2 system provides a novel strategy for biofilm elimination, and other applications utilizing oxidative breakdown.Hydrogen peroxide (H2O2) is a ``green chemical'' that has various cleaning and disinfectant uses, including as an anti-bacterial agent for hygienic and medical treatments. However, its efficacy is limited against biofilm-producing bacteria, because of poor penetration into the protective, organic matrix. Here we show new applications for ferromagnetic nanoparticles (Fe3O4, MNPs) with peroxidase-like activity in potentiating the efficacy of H2O2 in biofilm degradation and prevention. Our data show that MNPs enhanced oxidative cleavage of biofilm components (model nucleic acids, proteins, and oligosaccharides) in the presence of H2O2. When challenged with live, biofilm-producing bacteria, the MNP-H2O2 system efficiently broke down the existing biofilm and prevented new biofilms from forming, killing both planktonic bacteria and those within the biofilm. By enhancing oxidative cleavage of various substrates, the MNP-H2O2 system provides a novel

  14. Concepts of Ideal and Nonideal Explosives.

    DTIC Science & Technology

    1981-12-01

    Akst and J. Hershkowitz, "Explosive Performance Modification by Cosolidifaction of Ammonium Nitrate with Fuels ," Technical Report 4987, Picatinny...explosives Equations of state Diameter effect Ammonium nitrate 20. ASSrRACr (ca’mes r w re t N netwezy ad identity by block number) The purpose of...this report is to stimulate discussion on the nonideality of ammonium nitrate and its composite explosives. The concept of ideal and non- ideal

  15. Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.

    PubMed Central

    Fu, D J; McLaughlin, L W

    1992-01-01

    Eight modified ribozymes of 19 residues have been prepared with individual purine amino or hydroxyl groups excised. The modified ribozymes were chemically synthesized with the substitution of a single 2'-deoxyadenosine, 2'-deoxyguanosine, inosine, or purine riboside for residues G10, A11, G13, or A14. Five of the modified ribozymes cleaved the 24-mer substrate with little change in rate as monitored by simple first-order kinetics. However, deletion of the 2-amino group at G10 (replacement with inosine) or deletion of either of the 2'-hydroxyls at G10 or G13 (replacement with 2'-deoxyguanosine) resulted in ribozymes with a drastic decrease in cleavage efficiency. Increasing the concentration of the Mg2+ cofactor from 10 mM to 50 mM significantly enhanced cleavage efficiency by these three derivatives. Steady-state kinetic assays for these three ribozymes indicated that the modifications result in both an increase in Km and a decrease in kcat. These results suggest that the exocyclic amino group at-G10 and the hydroxyls at G10 and G13 are important both for ribozyme-substrate binding and for the Mg(2+)-catalyzed cleavage reaction. PMID:1570323

  16. [Research progress of bonding strength between porcelain veneer and enamel].

    PubMed

    Cheng, Hong; Zhang, Fu-qiang

    2014-02-01

    Porcelain veneer had gained more and more attention in dental clinical applications due to its advantages such as good esthetic effects and minor invasiveness. The reliable and consistent adhesive bonding were the key to success. The enamel which featured high mineralization and low moisture would be the ideal bonding part for porcelain veneer. This article was aimed to summarize the research progress regarding to those factors that might had effect on the bonding strength between the porcelain veneer and the enamel including the restoration types of resin adhesives and bonding surface preparations.

  17. Impact of non-ideal analyte behavior on the separation of protein aggregates by asymmetric flow field-flow fractionation.

    PubMed

    Boll, Björn; Josse, Lena; Heubach, Anja; Hochenauer, Sophie; Finkler, Christof; Huwyler, Jörg; Koulov, Atanas V

    2018-04-25

    Asymmetric flow field-flow fractionation is a valuable tool for the characterization of protein aggregates in biotechnology owing to its broad size range and unique separation principle. However, in practice asymmetric flow field-flow fractionation is non-trivial to use due to the major deviations from theory and the influence on separation by various factors that are not fully understood. Here we report methods to assess the non-ideal effects that influence asymmetric flow field-flow fractionation separation and for the first time identify experimentally the main factors that impact it. Furthermore, we propose new approaches to minimize such non-ideal behavior, showing that by adjusting the mobile phase composition (pH and ionic strength) the resolution of asymmetric flow field-flow fractionation separation can be drastically improved. Additionally, we propose a best practice method for new proteins. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  18. Metal-organic framework catalysts for selective cleavage of aryl-ether bonds

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Allendorf, Mark D.; Stavila, Vitalie

    The present invention relates to methods of employing a metal-organic framework (MOF) as a catalyst for cleaving chemical bonds. In particular instances, the MOF results in selective bond cleavage that results in hydrogenolyzis. Furthermore, the MOF catalyst can be reused in multiple cycles. Such MOF-based catalysts can be useful, e.g., to convert biomass components.

  19. Ideal and Nonideal Reasoning in Educational Theory

    ERIC Educational Resources Information Center

    Jaggar, Alison M.

    2015-01-01

    The terms "ideal theory" and "nonideal theory" are used in contemporary Anglophone political philosophy to identify alternative methodological approaches for justifying normative claims. Each term is used in multiple ways. In this article Alison M. Jaggar disentangles several versions of ideal and nonideal theory with a view to…

  20. Idealized cultural beliefs about gender: implications for mental health.

    PubMed

    Mahalingam, Ramaswami; Jackson, Benita

    2007-12-01

    In this paper, we examined the relationship between culture-specific ideals (chastity, masculinity, caste beliefs) and self-esteem, shame and depression using an idealized cultural model proposed by Mahalingam (2006, In: Mahalingam R (ed) Cultural psychology of immigrants. Lawrence Erlbaum, Mahwah, NJ, pp 1-14). Participants were from communities with a history of extreme male-biased sex ratios in Tamilnadu, India (N = 785). We hypothesized a dual-process model of self-appraisals suggesting that achieving idealized cultural identities would increase both self-esteem and shame, with the latter leading to depression, even after controlling for key covariates. We tested this using structural equation modeling. The proposed idealized cultural identities model had an excellent fit (CFI = 0.99); the effect of idealized identities on self-esteem, shame and depression differed by gender. Idealized beliefs about gender relate to psychological well-being in gender specific ways in extreme son preference communities. We discuss implications of these findings for future research and community-based interventions.

  1. Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

    PubMed

    Kristensen, Thea; Normann, Preben; Gullberg, Maria; Fahnøe, Ulrik; Polacek, Charlotta; Rasmussen, Thomas Bruun; Belsham, Graham J

    2017-03-01

    The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

  2. Pyrexia's effect on the CBG-cortisol thermocouple, rather than CBG cleavage, elevates the acute free cortisol response to TNF-α in humans.

    PubMed

    Nenke, Marni Anne; Nielsen, Signe Tellerup; Lehrskov, Louise Lang; Lewis, John Goodwyn; Rankin, Wayne; Møller, Kirsten; Torpy, David James

    2017-03-01

    Corticosteroid-binding globulin (CBG) cleavage promotes local cortisol delivery in inflammation. Enzymatic cleavage of high-affinity CBG to low-affinity CBG (haCBG to laCBG) occurs at inflammatory sites and is now measurable in vivo; however, the time kinetics of haCBG depletion following an inflammatory stimulus is unknown. Hence our aim was to determine the immediate effect of the key pro-inflammatory cytokine TNF-α on CBG levels and cleavage. We performed a crossover study of 12 healthy males receiving a TNF-α versus saline infusion, measuring total CBG, haCBG, laCBG and free and total cortisol hourly for 6 h. There was no change in total CBG or haCBG levels in the first 6 h of inflammation between the groups, suggesting that CBG cleavage is not activated nor is hepatic CBG production affected by TNF-α in this time frame. There was an early increase in the ratio of free:total cortisol, in association with pyrexia. This accords with data indicating that CBG acts a thermocouple in vivo, increasing free cortisol levels independent of elastase-driven cleavage.

  3. Effect of the timing of first cleavage on in vitro developmental potential of nuclear-transferred bovine oocytes receiving cumulus and fibroblast cells.

    PubMed

    Amarnath, Dasari; Kato, Yoko; Tsunoda, Yukio

    2007-06-01

    The aim of the present study was to examine whether cumulus and fibroblast cell nuclear-transferred oocytes, which have high and low potential to develop into normal calves, respectively, are different in terms of in their patterns of timing of first cleavage and in their relationships between timing of first cleavage and in vitro developmental potential. The timing of first cleavage was similar in both types of nuclear-transferred and in vitro fertilized oocytes. More than 86% of the oocytes cleaved within 24 h after activation or in vitro fertilization; these oocytes contributed to more than 98% of the total number of blastocysts in all three groups. The potential of oocytes that cleaved at different intervals to develop into blastocysts differed among the groups. The developmental potential of the cumulus cell nuclear-transferred oocytes and in vitro fertilized oocytes decreased with the increase in time required for cleavage. Fibroblast cell nuclear-transferred oocytes that cleaved at 20 h, an intermediate cleaving time, had higher potential to develop into blastocysts. The results of the present study suggest that the type of donor nucleus used for nuclear transfer affects the timing of first cleavage.

  4. Susceptibility for thin ideal media and eating styles.

    PubMed

    Anschutz, Doeschka J; Engels, Rutger C M E; Van Strien, Tatjana

    2008-03-01

    This study examined the relations between susceptibility for thin ideal media and restrained, emotional and external eating, directly and indirectly through body dissatisfaction. Thin ideal media susceptibility, body dissatisfaction and eating styles were measured in a sample of 163 female students. Structural equation modelling was used for analyses, controlling for BMI. Higher susceptibility for thin ideal media was directly related to higher scores on all eating styles, and indirectly related to higher restrained and emotional eating through elevated levels of body dissatisfaction. So, thin ideal media susceptibility was not only related to restraint through body dissatisfaction, but also directly. Emotional eaters might be more vulnerable for negative affect, whereas external eaters might be more sensitive to external cues in general.

  5. Metal ion-promoted cleavage of nucleoside diphosphosugars: a model for reactions of phosphodiester bonds in carbohydrates.

    PubMed

    Dano, Meisa; Elmeranta, Marjukka; Hodgson, David R W; Jaakkola, Juho; Korhonen, Heidi; Mikkola, Satu

    2015-12-01

    Cleavage of five different nucleoside diphosphosugars has been studied in the presence of Cu(2+) and Zn(2+) complexes. The results show that metal ion catalysts promote the cleavage via intramolecular transesterification whenever a neighbouring HO group can adopt a cis-orientation with respect to the phosphate. The HO group attacks the phosphate and two monophosphate products are formed. If such a nucleophile is not available, Cu(2+) complexes are able to promote a nucleophilic attack of an external nucleophile, e.g. a water molecule or metal ion coordinated HO ligand, on phosphate. With the Zn(2+) complex, this was not observed.

  6. Thin idealization and causal attributions mediate the association between culture and obesity stereotypes: An examination of Chinese and American adolescents.

    PubMed

    Klaczynski, Paul A; Felmban, Wejdan S

    2018-05-28

    Few studies have examined age or cultural differences in the stereotypes adolescents have of persons with obesity. The present research explored the hypotheses that American adolescents have more negative obesity stereotypes than Chinese adolescents and that the effects of culture are mediated by weight attributions and thin idealization. Participants (N = 335; 181 female; M age = 14.83 years, SD = 1.57 years) completed measures of thin idealization and causal attributions and made generalizations from and attributions of stereotypical personality characteristics to obese figures. Not only did stereotypes differ between countries, but generalizations of negative characteristics from obese figures increased with age. In addition, American adolescents more firmly endorsed the 'thin ideal' and were more likely to attribute obesity to internal causes that Chinese adolescents. As anticipated, between-country differences in stereotyping were mediated by thin idealization and causal attributions. Findings are discussed in terms of the 'doctrine of the mean', social identity theory, and dual-process theories. Statement of Contribution The development of obesity stereotypes has been the subject of a number of recent studies. Although scarce, research on adolescents' obesity stereotypes indicates that the strength of these stereotypes increases with age and that these increases are mediated by thin idealization and causal attributions. The current research adds to this growing literature that differences between Chinese adolescents' and American adolescents' obesity stereotypes - in terms of the assignment of stereotypical traits to people with obesity and the generalization of negative traits from an individual person with obesity to people with obesity as a group - are mediated by thin idealization and attributions about obesity's causes. The research also indicates that (1) age differences in obesity stereotyping vary as a function of the method used to measure

  7. Promoting Spiritual Ideals through Design Thinking in Public Schools

    ERIC Educational Resources Information Center

    Tan, Charlene; Wong, Yew-Leong

    2012-01-01

    Against a backdrop of the debates on religious education in public or state schools, we argue for the introduction of "spiritual ideals" into the public school curriculum. We distinguish our notion of spiritual ideals from "religious ideals" as conceptualised by De Ruyter and Merry. While we agree with De Ruyter and Merry that…

  8. A new trinuclear complex of platinum and iron efficiently promotes cleavage of plasmid DNA.

    PubMed Central

    Lempers, E L; Bashkin, J S; Kostić, N M

    1993-01-01

    The compound [[Pt(trpy)]2Arg-EDTA]+ is synthesized in five steps, purified, and characterized by 1H, 13C, and 195Pt NMR spectroscopy, mass spectrometry, UV-vis spectrophotometry, and elemental analysis. The binuclear [[(Pt(trpy)]2Arg]3+ moiety binds to double-stranded DNA, and the chelating EDTA moiety holds metal cations. In the presence of ferrous ions and the reductant dithiothreitol, the new compound cleaves DNA. It cleaves a single strand in the pBR322 plasmid nearly as efficiently as methidiumrpropyl-EDTA (MPE), and it cleaves a restriction fragment of the XP10 plasmid nonselectively and more efficiently than [Fe(EDTA)]2-. The mechanism of cleavage was studied in control experiments involving different transition-metal ions, superoxide dismutase, catalase, glucose oxidase with glucose, metal-sequestering agents, and deaeration. These experiments indicate that adventitious iron and copper ions, superoxide anion, and hydrogen peroxide are not involved and that dioxygen is required. The cleavage apparently is done by hydroxyl radicals generated in the vicinity of the DNA molecule. The reagent [[Pt(trypy)]2Arg-EDTA]+ differs from methidiumpropyl-EDTA in not containing an intercalator. This difference in binding modes between the binuclear platinum(II) complex and the planar heterocycle may cause useful differences between the two reagents in cleavage of nucleic acids. Images PMID:8493109

  9. Proton-driven amide bond-cleavage pathways of gas-phase peptide ions lacking mobile protons.

    PubMed

    Bythell, Benjamin J; Suhai, Sándor; Somogyi, Arpád; Paizs, Béla

    2009-10-07

    The mobile proton model (Dongre, A. R., Jones, J. L., Somogyi, A. and Wysocki, V. H. J. Am. Chem. Soc. 1996, 118 , 8365-8374) of peptide fragmentation states that the ionizing protons play a critical role in the gas-phase fragmentation of protonated peptides upon collision-induced dissociation (CID). The model distinguishes two classes of peptide ions, those with or without easily mobilizable protons. For the former class mild excitation leads to proton transfer reactions which populate amide nitrogen protonation sites. This enables facile amide bond cleavage and thus the formation of b and y sequence ions. In contrast, the latter class of peptide ions contains strongly basic functionalities which sequester the ionizing protons, thereby often hindering formation of sequence ions. Here we describe the proton-driven amide bond cleavages necessary to produce b and y ions from peptide ions lacking easily mobilizable protons. We show that this important class of peptide ions fragments by different means from those with easily mobilizable protons. We present three new amide bond cleavage mechanisms which involve salt-bridge, anhydride, and imine enol intermediates, respectively. All three new mechanisms are less energetically demanding than the classical oxazolone b(n)-y(m) pathway. These mechanisms offer an explanation for the formation of b and y ions from peptide ions with sequestered ionizing protons which are routinely fragmented in large-scale proteomics experiments.

  10. Childhood Lifestyle and Clinical Determinants of Adult Ideal

    PubMed Central

    Laitinen, Tomi T.; Pahkala, Katja; Venn, Alison; Woo, Jessica G; Oikonen, Mervi; Dwyer, Terence; Mikkilä, Vera; Hutri-Kähönen, Nina; Smith, Kylie J.; Gall, Seana L.; Morrison, John A.; Viikari, Jorma S.A.; Raitakari, Olli T.; Magnussen, Costan G.; Juonala, Markus

    2013-01-01

    Background The American Heart Association recently defined ideal cardiovascular health by simultaneous presence of seven health behaviors and factors. The concept is associated with cardiovascular disease incidence, and cardiovascular disease and all-cause mortality. To effectively promote ideal cardiovascular health already early in life, childhood factors predicting future ideal cardiovascular health should be investigated. Our aim was thus to comprehensively explore childhood determinants of adult ideal cardiovascular health in population based cohorts from three continents. Methods The sample comprised a total of 4409 participants aged 3–19 years at baseline from the Cardiovascular Risk in Young Finns Study (YFS; N=1883) from Finland, Childhood Determinants of Adult Health Study (CDAH; N=1803) from Australia and Princeton Follow-up Study (PFS; N=723) from the United States. Participants were re-examined 19–31 years later when aged 30–48 years. Results In multivariable analyses, independent childhood predictors of adult ideal cardiovascular health were family socioeconomic status (P<0.01; direct association) and BMI (P<0.001; inverse association) in all cohorts. In addition, blood pressure (P=0.007), LDL-cholesterol (P<0.001) and parental smoking (P=0.006) in the YFS, and own smoking (P=0.001) in CDAH were inversely associated with future ideal cardiovascular health. Conclusions Among several lifestyle and clinical indicators studied, higher family socioeconomic status and non-smoking (parental/own) in childhood independently predict ideal cardiovascular health in adulthood. As atherosclerotic cardiovascular diseases are rooted in childhood, our findings suggest that special attention could be paid to children who are from low socioeconomic status families, and who smoke or whose parents smoke, to prevent cardiovascular disease morbidity and mortality. PMID:24075574

  11. 100 M.a. remagnetization as a dating tool for deformation and cleavage in the Central High Atlas (Morocco)

    NASA Astrophysics Data System (ADS)

    Calvin, P.; Casas, A. M.; Villalain, J. J.; Moussaid, B.

    2015-12-01

    The High Atlas is an intracontinental chain developed as a result of the inversion of Mesozoic basins during the Cenozoic. Its structure is characterized by ENE-WSW tight anticlines limited by wide synclines. In the central sector of the chain, a pervasive tectonic foliation affects Jurassic limestones and marls. There is a controversy about the age of this tectonic foliation and its relationship with either a Late Jurassic or Cenozoic compressional events. The Jurassic carbonates of the Central High Atlas (CHA) show a widespread syntectonic remagnetization carried by magnetite and dated at 100 M.a. by comparing the paleomagnetic direction obtained by small circle intersection (SCI) method with the apparent polar wander path in NW Africa coordinates. Once the 100 M.a. paleomagnetic direction is known, the obtained paleomagnetic direction remagnetization in each site can be used to restore the bedding at the time of the acquisition. In each site, the in situ mean direction of remagnetization defines a small circle (SC) in a complete rotation about the strike of the bedding. This SC gives all possible original directions of the magnetization and if all deformation events are coaxial and without vertical rotation (as it is the case in the study area) all the SC contains the 100 M.a. expected direction. Then, the angle between the 100 M.a. and the in situ paleomagnetic direction along the small circle, equals the angle of rotation of each bed to ultimately find their dips (paleodip) at 100 M.a. Since the consistency between folding and cleavage can be examined from their geometrical relationship, and bedding can be restored to its 100 M.a. geometry (paleodips obtained from paleomagnetic analysis), two end-members exist for the different examined folds: (i) cleavage is consistent with present-day bedding orientation and attitude of bedding was acquired after remagnetization (Cenozoic cleavage), (ii) cleavage is consistent with bedding, but dip of bedding was acquired

  12. Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients

    PubMed Central

    2012-01-01

    Objectives This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. Study design We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Results Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups. Conclusions In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic

  13. Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients.

    PubMed

    Tong, Guo Qing; Cao, Shan Ren; Wu, Xun; Zhang, Jun Qiang; Cui, Ji; Heng, Boon Chin; Ling, Xiu Feng

    2012-10-05

    This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups. In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic Chinese patients.

  14. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

    PubMed

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter; Vidal-Melgosa, Silvia; Yan, Kok-Phen; Fangel, Jonatan Ulrik; Meyer, Anne S; Kirpekar, Finn; Willats, William G; Mikkelsen, Jørn D

    2014-12-01

    Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

  15. On the union of graded prime ideals

    NASA Astrophysics Data System (ADS)

    Uregen, Rabia Nagehan; Tekir, Unsal; Hakan Oral, Kursat

    2016-01-01

    In this paper we investigate graded compactly packed rings, which is defined as; if any graded ideal I of R is contained in the union of a family of graded prime ideals of R, then I is actually contained in one of the graded prime ideals of the family. We give some characterizations of graded compactly packed rings. Further, we examine this property on h - Spec(R). We also define a generalization of graded compactly packed rings, the graded coprimely packed rings. We show that R is a graded compactly packed ring if and only if R is a graded coprimely packed ring whenever R be a graded integral domain and h - dim R = 1.

  16. Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

    PubMed Central

    Kwong, M. Y.; Harris, R. J.

    1994-01-01

    Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

  17. Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A.

    PubMed Central

    Varro, A; Dockray, G J

    1993-01-01

    The precursor for the acid-stimulating hormone gastrin provides a useful model for studies of post-translational processing because defined sites of cleavage, amidation, sulphation and phosphorylation occur within a dodecapeptide sequence. The factors determining these post-translational processing events are still poorly understood. We have used brefeldin A, which disrupts transport from rough endoplasmic reticulum to the Golgi complex, to examine the mechanisms of cleavage, phosphorylation and sulphation of rat progastrin-derived peptides. Biosynthetic products were detected after immunoprecipitation using antibodies specific for the extreme C-terminus of progastrin, followed by reversed-phase and ion-exchange h.p.l.c. Gastrin cells incorporated [3H]tyrosine, [32P]phosphate and [35S]sulphate into both progastrin and its extreme C-terminal tryptic (nona-) peptide. Ion-exchange chromatography resolved four forms of the C-terminal tryptic fragment of progastrin which differed in whether they were phosphorylated at Ser96, sulphated at Tyr103, both or neither. The specific activity of [3H]tyrosine in the peak that was both phosphorylated and sulphated was higher than in the others. Brefeldin A inhibited the appearance of [3H]tyrosine-labelled C-terminal tryptic fragment but there was an accumulation of labelled progastrin and a peptide corresponding to the C-terminal 46 residues of progastrin. Brefeldin A also inhibited incorporation of 32P and 35S into both progastrin and its C-terminal fragment. Thus phosphorylation of Ser96, sulphation of Tyr103 and cleavage at Arg94-Arg95 depend on passage of newly synthesized progastrin along the secretory pathway; as brefeldin A is thought to act proximal to the trans-Golgi, these processing steps would appear to occur distal to this point. The data also indicate that the stores of unphosphorylated C-terminal tryptic fragment are not available for phosphorylation, implying that this modification occurs proximal to the secretory

  18. Why Education in Public Schools Should Include Religious Ideals

    ERIC Educational Resources Information Center

    de Ruyter, Doret J.; Merry, Michael S.

    2009-01-01

    This article aims to open a new line of debate about religion in public schools by focusing on religious ideals. The article begins with an elucidation of the concept "religious ideals" and an explanation of the notion of reasonable pluralism, in order to be able to explore the dangers and positive contributions of religious ideals and their…

  19. Mechanism of endonuclease cleavage by the HigB toxin

    PubMed Central

    Schureck, Marc A.; Repack, Adrienne; Miles, Stacey J.; Marquez, Jhomar; Dunham, Christine M.

    2016-01-01

    Bacteria encode multiple type II toxin–antitoxin modules that cleave ribosome-bound mRNAs in response to stress. All ribosome-dependent toxin family members structurally characterized to date adopt similar microbial RNase architectures despite possessing low sequence identities. Therefore, determining which residues are catalytically important in this specialized RNase family has been a challenge in the field. Structural studies of RelE and YoeB toxins bound to the ribosome provided significant insights but biochemical experiments with RelE were required to clearly demonstrate which residues are critical for acid-base catalysis of mRNA cleavage. Here, we solved an X-ray crystal structure of the wild-type, ribosome-dependent toxin HigB bound to the ribosome revealing potential catalytic residues proximal to the mRNA substrate. Using cell-based and biochemical assays, we further determined that HigB residues His54, Asp90, Tyr91 and His92 are critical for activity in vivo, while HigB H54A and Y91A variants have the largest effect on mRNA cleavage in vitro. Comparison of X-ray crystal structures of two catalytically inactive HigB variants with 70S-HigB bound structures reveal that HigB active site residues undergo conformational rearrangements likely required for recognition of its mRNA substrate. These data support the emerging concept that ribosome-dependent toxins have diverse modes of mRNA recognition. PMID:27378776

  20. Macro-mesoscopic Fracture and Strength Character of Pre-cracked Granite Under Stress Relaxation Condition

    NASA Astrophysics Data System (ADS)

    Liu, Junfeng; Yang, Haiqing; Xiao, Yang; Zhou, Xiaoping

    2018-05-01

    The fracture characters are important index to study the strength and deformation behavior of rock mass in rock engineering. In order to investigate the influencing mechanism of loading conditions on the strength and macro-mesoscopic fracture character of rock material, pre-cracked granite specimens are prepared to conduct a series of uniaxial compression experiments. For parts of the experiments, stress relaxation tests of different durations are also conducted during the uniaxial loading process. Furthermore, the stereomicroscope is adopted to observe the microstructure of the crack surfaces of the specimens. The experimental results indicate that the crack surfaces show several typical fracture characters in accordance with loading conditions. In detail, some cleavage fracture can be observed under conventional uniaxial compression and the fractured surface is relatively rough, whereas as stress relaxation tests are attached, relative slip trace appears between the crack faces and some shear fracture starts to come into being. Besides, the crack faces tend to become smoother and typical terrace structures can be observed in local areas. Combining the macroscopic failure pattern of the specimens, it can be deduced that the duration time for the stress relaxation test contributes to the improvement of the elastic-plastic strain range as well as the axial peak strength for the studied material. Moreover, the derived conclusion is also consistent with the experimental and analytical solution for the pre-peak stage of the rock material. The present work may provide some primary understanding about the strength character and fracture mechanism of hard rock under different engineering environments.

  1. HIV-1 protease cleavage site prediction based on two-stage feature selection method.

    PubMed

    Niu, Bing; Yuan, Xiao-Cheng; Roeper, Preston; Su, Qiang; Peng, Chun-Rong; Yin, Jing-Yuan; Ding, Juan; Li, HaiPeng; Lu, Wen-Cong

    2013-03-01

    Knowledge of the mechanism of HIV protease cleavage specificity is critical to the design of specific and effective HIV inhibitors. Searching for an accurate, robust, and rapid method to correctly predict the cleavage sites in proteins is crucial when searching for possible HIV inhibitors. In this article, HIV-1 protease specificity was studied using the correlation-based feature subset (CfsSubset) selection method combined with Genetic Algorithms method. Thirty important biochemical features were found based on a jackknife test from the original data set containing 4,248 features. By using the AdaBoost method with the thirty selected features the prediction model yields an accuracy of 96.7% for the jackknife test and 92.1% for an independent set test, with increased accuracy over the original dataset by 6.7% and 77.4%, respectively. Our feature selection scheme could be a useful technique for finding effective competitive inhibitors of HIV protease.

  2. 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage.

    PubMed

    Barlos, K; Chatzi, O; Gatos, D; Stavropoulos, G

    1991-06-01

    The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp, Met, and Tyr.

  3. Recent Advances in Ring-Opening Functionalization of Cycloalkanols by C-C σ-Bond Cleavage.

    PubMed

    Wu, Xinxin; Zhu, Chen

    2018-06-01

    Cycloalkanols prove to be privileged precursors for the synthesis of distally substituted alkyl ketones and polycyclic aromatic hydrocarbons (PAHs) by virtue of cleavage of their cyclic C-C bonds. Direct functionalization of cyclobutanols to build up other chemical bonds (e. g., C-F, C-Cl, C-Br, C-N, C-S, C-Se, C-C, etc.) has been achieved by using the ring-opening strategy. Mechanistically, the C-C cleavage of cyclobutanols can be involved in two pathways: (a) transition-metal catalyzed β-carbon elimination; (b) radical-mediated 'radical clock'-type ring opening. The recent advances of our group for the ring-opening functionalization of tertiary cycloalkanols are described in this account. © 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Idealization of the analyst by the young adult.

    PubMed

    Chused, J F

    1987-01-01

    Idealization is an intrapsychic process that serves many functions. In addition to its use defensively and for gratification of libidinal and aggressive drive derivatives, it can contribute to developmental progression, particularly during late adolescence and young adulthood. During an analysis, it is important to recognize all the determinants of idealization, including those related to the reworking of developmental conflicts. If an analyst understands idealization solely as a manifestation of pathology, he may interfere with his patient's use of it for the development of autonomous functioning.

  5. ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site.

    PubMed

    Abbruzzese, Genevieve; Becker, Sarah F; Kashef, Jubin; Alfandari, Dominique

    2016-07-15

    The cranial neural crest (CNC) is a highly motile population of cells that is responsible for forming the face and jaw in all vertebrates and perturbing their migration can lead to craniofacial birth defects. Cell motility requires a dynamic modification of cell-cell and cell-matrix adhesion. In the CNC, cleavage of the cell adhesion molecule cadherin-11 by ADAM13 is essential for cell migration. This cleavage generates a shed extracellular fragment of cadherin-11 (EC1-3) that possesses pro-migratory activity via an unknown mechanism. Cadherin-11 plays an important role in modulating contact inhibition of locomotion (CIL) in the CNC to regulate directional cell migration. Here, we show that while the integral cadherin-11 requires the homophilic binding site to promote CNC migration in vivo, the EC1-3 fragment does not. In addition, we show that increased ADAM13 activity or expression of the EC1-3 fragment increases CNC invasiveness in vitro and blocks the repulsive CIL response in colliding cells. This activity requires the presence of an intact homophilic binding site on the EC1-3 suggesting that the cleavage fragment may function as a competitive inhibitor of cadherin-11 adhesion in CIL but not to promote cell migration in vivo. Copyright © 2015. Published by Elsevier Inc.

  6. ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site

    PubMed Central

    Kashef, Jubin; Alfandari, Dominique

    2015-01-01

    The cranial neural crest (CNC) is a highly motile population of cells that is responsible for forming the face and jaw in all vertebrates and perturbing their migration can lead to craniofacial birth defects. Cell motility requires a dynamic modification of cell–cell and cell-matrix adhesion. In the CNC, cleavage of the cell adhesion molecule cadherin-11 by ADAM13 is essential for cell migration. This cleavage generates a shed extracellular fragment of cadherin-11 (EC1-3) that possesses pro-migratory activity via an unknown mechanism. Cadherin-11 plays an important role in modulating contact inhibition of locomotion (CIL) in the CNC to regulate directional cell migration. Here, we show that while the integral cadherin-11 requires the homophilic binding site to promote CNC migration in vivo, the EC1-3 fragment does not. In addition, we show that increased ADAM13 activity or expression of the EC1-3 fragment increases CNC invasiveness in vitro and blocks the repulsive CIL response in colliding cells. This activity requires the presence of an intact homophilic binding site on the EC1-3 suggesting that the cleavage fragment may function as a competitive inhibitor of cadherin-11 adhesion in CIL but not to promote cell migration in vivo. PMID:26206614

  7. Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

    PubMed

    De Lorenzi, Valentina; Sarra Ferraris, Gian Maria; Madsen, Jeppe B; Lupia, Michela; Andreasen, Peter A; Sidenius, Nicolai

    2016-07-01

    Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process. © 2016 The Authors.

  8. The Echinoid Mitotic Gradient: Effect of Cell Size on the Micromere Cleavage Cycle

    PubMed Central

    Langelan Duncan, Rosalie E.; Whiteley, Arthur H.

    2012-01-01

    SUMMARY Like other euechinoids, the fertilized eggs of the sand dollar Dendraster excentricus proceed through cleavages that produce a pattern of macromeres, mesomeres, and micromeres at the 4th division. The 8 cells of the macro-mesomere lineage proceed through 6 additional cleavages before hatching. At the fifth overall division, the 4 micromeres produce a lineage of large micromeres that will divide 3 additional times, and a lineage of small micromeres that will divide once more before hatching. Irrespective of lineage, the length of the cell cycles is closely related to the size of the blastomere; cells of the same size have the same cell cycle time. A consequence is that at the fourth cleavage, there is a gradient of mitotic activity from the fastest dividers at the animal pole and the slowest cleacing micromeres at the vegetal pole. By the time of hatching, which is the 10th division of meso-macromeres, all cells are the same small size, the metachronic pattern of division gives way to asynchrony, and the mitotic gradient along the polar axis is lost. Experimental pre-exposure to sodium dodecyl sulfate (SDS), however, blocks the appearance of the gradients in cell size, the mitotic gradient, and the differential in cell cycle times. It is proposed that the mitotic gradients, cell cycle times, and attainment of a state of asynchrony are functions of cell size. Developmental consequences of the transition are large, and include coordinated activation of transcriptions, synthesis of new patterns of proteins, alterations of metabolism, and onset of morphogenesis. PMID:22006441

  9. Copper-Catalyzed Oxidative Reaction of β-Keto Sulfones with Alcohols via C-S Bond Cleavage: Reaction Development and Mechanism Study.

    PubMed

    Du, Bingnan; Wang, Wenmin; Wang, Yang; Qi, Zhenghang; Tian, Jiaqi; Zhou, Jie; Wang, Xiaochen; Han, Jianlin; Ma, Jing; Pan, Yi

    2018-02-16

    A Cu-catalyzed cascade oxidative radical process of β-keto sulfones with alcohols has been achieved by using oxygen as an oxidant. In this reaction, β-keto sulfones were converted into sulfinate esters under the oxidative conditions via cleavage of C-S bond. Experimental and computational studies demonstrate that a new pathway is involved in this reaction, which proceeds through the formation of the key four-coordinated Cu II intermediate, O-O bond homolysis induced C-S bond cleavage and Cu-catalyzed esterification to form the final products. This reaction provides a new strategy to sulfonate esters and enriches the research content of C-S bond cleavage and transformations. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Challenges in Determining Intrinsic Viscosity Under Low Ionic Strength Solution Conditions.

    PubMed

    Pindrus, Mariya A; Shire, Steven J; Yadav, Sandeep; Kalonia, Devendra S

    2017-04-01

    To determine the intrinsic viscosity of several monoclonal antibodies (mAbs) under varying pH and ionic strength solution conditions. An online viscosity detector attached to HPLC (Viscotek®) was used to determine the intrinsic viscosity of mAbs. The Ross and Minton equation was used for viscosity prediction at high protein concentrations. Bulk viscosity was determined by a Cambridge viscometer. At 15 mM ionic strength, intrinsic viscosity of the mAbs determined by the single-point approach varied from 5.6 to 6.4 mL/g with changes in pH. High ionic strength did not significantly alter intrinsic viscosity, while a significant increase (up to 24.0 mL/g) was observed near zero mM. No difference in bulk viscosity of mAb3 was observed around pH 6 as a function of ionic strength. Data analysis revealed that near zero mM ionic strength limitations of the single-point technique result in erroneously high intrinsic viscosity. Intrinsic viscosity is a valuable tool that can be used to model baseline viscosity at higher protein concentrations. However, it is not predictive of solution non-ideality at higher protein concentrations. Furthermore, breakdown of numerous assumptions limits the applicability of experimental techniques near zero mM ionic strength conditions. For molecules and conditions studied, the single-point approach produced reliable intrinsic viscosity results at 15 mM. However, this approach must be used with caution near zero mM ionic strength. Data analysis can be used to reveal whether determined intrinsic viscosity is reliable or erroneously high.

  11. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Spannaus, Ralf; Bodem, Jochen, E-mail: Jochen.Bodem@vim.uni-wuerzburg.de

    2014-04-15

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies.more » The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.« less

  12. Impact of physical parameterizations on idealized tropical cyclones in the Community Atmosphere Model

    NASA Astrophysics Data System (ADS)

    Reed, K. A.; Jablonowski, C.

    2011-02-01

    This paper explores the impact of the physical parameterization suite on the evolution of an idealized tropical cyclone within the National Center for Atmospheric Research's (NCAR) Community Atmosphere Model (CAM). The CAM versions 3.1 and 4 are used to study the development of an initially weak vortex in an idealized environment over a 10-day simulation period within an aqua-planet setup. The main distinction between CAM 3.1 and CAM 4 lies within the physical parameterization of deep convection. CAM 4 now includes a dilute plume Convective Available Potential Energy (CAPE) calculation and Convective Momentum Transport (CMT). The finite-volume dynamical core with 26 vertical levels in aqua-planet mode is used at horizontal grid spacings of 1.0°, 0.5° and 0.25°. It is revealed that CAM 4 produces stronger and larger tropical cyclones by day 10 at all resolutions, with a much earlier onset of intensification when compared to CAM 3.1. At the highest resolution CAM 4 also accounts for changes in the storm's vertical structure, such as an increased outward slope of the wind contours with height, when compared to CAM 3.1. An investigation concludes that the new dilute CAPE calculation in CAM 4 is largely responsible for the changes observed in the development, strength and structure of the tropical cyclone.

  13. Post-translational cleavage of Hv1 in human sperm tunes pH- and voltage-dependent gating.

    PubMed

    Berger, Thomas K; Fußhöller, David M; Goodwin, Normann; Bönigk, Wolfgang; Müller, Astrid; Dokani Khesroshahi, Nasim; Brenker, Christoph; Wachten, Dagmar; Krause, Eberhard; Kaupp, U Benjamin; Strünker, Timo

    2017-03-01

    In human sperm, proton flux across the membrane is controlled by the voltage-gated proton channel Hv1. We show that sperm harbour both Hv1 and an N-terminally cleaved isoform termed Hv1Sper. The pH-control of Hv1Sper and Hv1 is distinctively different. Hv1Sper and Hv1 can form heterodimers that combine features of both constituents. Cleavage and heterodimerization of Hv1 might represent an adaptation to the specific requirements of pH control in sperm. In human sperm, the voltage-gated proton channel Hv1 controls the flux of protons across the flagellar membrane. Here, we show that sperm harbour Hv1 and a shorter isoform, termed Hv1Sper. Hv1Sper is generated from Hv1 by removal of 68 amino acids from the N-terminus by post-translational proteolytic cleavage. The pH-dependent gating of the channel isoforms is distinctly different. In both Hv1 and Hv1Sper, the conductance-voltage relationship is determined by the pH difference across the membrane (∆pH). However, simultaneous changes in intracellular and extracellular pH that leave ΔpH constant strongly shift the activation curve of Hv1Sper but not that of Hv1, demonstrating that cleavage of the N-terminus tunes pH sensing in Hv1. Moreover, we show that Hv1 and Hv1Sper assemble as heterodimers that combine features of both constituents. We suggest that cleavage and heterodimerization of Hv1 represents an adaptation to the specific requirements of pH control in sperm. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  14. Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta.

    PubMed

    Ilg, Andrea; Yu, Qiuju; Schaub, Patrick; Beyer, Peter; Al-Babili, Salim

    2010-08-01

    Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.

  15. Moral identity as moral ideal self: links to adolescent outcomes.

    PubMed

    Hardy, Sam A; Walker, Lawrence J; Olsen, Joseph A; Woodbury, Ryan D; Hickman, Jacob R

    2014-01-01

    The purposes of this study were to conceptualize moral identity as moral ideal self, to develop a measure of this construct, to test for age and gender differences, to examine links between moral ideal self and adolescent outcomes, and to assess purpose and social responsibility as mediators of the relations between moral ideal self and outcomes. Data came from a local school sample (Data Set 1: N = 510 adolescents; 10-18 years of age) and a national online sample (Data Set 2: N = 383 adolescents; 15-18 years of age) of adolescents and their parents. All outcome measures were parent-report (Data Set 1: altruism, moral personality, aggression, and cheating; Data Set 2: environmentalism, school engagement, internalizing, and externalizing), whereas other variables were adolescent-report. The 20-item Moral Ideal Self Scale showed good reliability, factor structure, and validity. Structural equation models demonstrated that, even after accounting for moral identity internalization, in Data Set 1 moral ideal self positively predicted altruism and moral personality and negatively predicted aggression, whereas in Data Set 2 moral ideal self positively predicted environmentalism and negatively predicted internalizing and externalizing symptoms. Further, purpose and social responsibility mediated most relations between moral ideal self and the outcomes in Data Set 2. Moral ideal self was unrelated to age but differentially predicted some outcomes across age. Girls had higher levels of moral ideal self than boys, although moral identity did not differentially predict outcomes between genders. Thus, moral ideal self is a salient element of moral identity and may play a role in morally relevant adolescent outcomes. PsycINFO Database Record (c) 2014 APA, all rights reserved.

  16. The Adam family metalloprotease Kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline

    PubMed Central

    Coleman, Hope A.; Labrador, Juan-Pablo; Chance, Rebecca K.; Bashaw, Greg J.

    2010-01-01

    Slits and their Roundabout (Robo) receptors mediate repulsive axon guidance at the Drosophila ventral midline and in the vertebrate spinal cord. Slit is cleaved to produce fragments with distinct signaling properties. In a screen for genes involved in Slit-Robo repulsion, we have identified the Adam family metalloprotease Kuzbanian (Kuz). Kuz does not regulate midline repulsion through cleavage of Slit, nor is Slit cleavage essential for repulsion. Instead, Kuz acts in neurons to regulate repulsion and Kuz can cleave the Robo extracellular domain in Drosophila cells. Genetic rescue experiments using an uncleavable form of Robo show that this receptor does not maintain normal repellent activity. Finally, Kuz activity is required for Robo to recruit its downstream signaling partner, Son of sevenless (Sos). These observations support the model that Kuz-directed cleavage is important for Robo receptor activation. PMID:20570941

  17. Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)*

    PubMed Central

    van der Post, Sjoerd; Subramani, Durai B.; Bäckström, Malin; Johansson, Malin E. V.; Vester-Christensen, Malene B.; Mandel, Ulla; Bennett, Eric P.; Clausen, Henrik; Dahlén, Gunnar; Sroka, Aneta; Potempa, Jan; Hansson, Gunnar C.

    2013-01-01

    The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation. PMID:23546879

  18. Enhancing Protein Disulfide Bond Cleavage by UV Excitation and Electron Capture Dissociation for Top-Down Mass Spectrometry.

    PubMed

    Wongkongkathep, Piriya; Li, Huilin; Zhang, Xing; Loo, Rachel R Ogorzalek; Julian, Ryan R; Loo, Joseph A

    2015-11-15

    The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in combination with ECD allowed the three disulfide bonds of insulin to be cleaved and the overall sequence coverage to be increased. For the larger sized ribonuclease A with four disulfide bonds, irradiation from an infrared laser (10.6 µm) to disrupt non-covalent interactions was combined with UV-activation to facilitate the cleavage of up to three disulfide bonds. Preferences for disulfide bond cleavage are dependent on protein structure and sequence. Disulfide bonds can reform if the generated radicals remain in close proximity. By varying the time delay between the UV-activation and the ECD events, it was determined that disulfide bonds reform within 10-100 msec after their UV-homolytic cleavage.

  19. Mechanisms of Bond Cleavage during Manganese Oxide and UV Degradation of Glyphosate: Results from Phosphate Oxygen Isotopes and Molecular Simulations.

    PubMed

    Jaisi, Deb P; Li, Hui; Wallace, Adam F; Paudel, Prajwal; Sun, Mingjing; Balakrishna, Avula; Lerch, Robert N

    2016-11-16

    Degradation of glyphosate in the presence of manganese oxide and UV light was analyzed using phosphate oxygen isotope ratios and density function theory (DFT). The preference of C-P or C-N bond cleavage was found to vary with changing glyphosate/manganese oxide ratios, indicating the potential role of sorption-induced conformational changes on the composition of intermediate degradation products. Isotope data confirmed that one oxygen atom derived solely from water was incorporated into the released phosphate during glyphosate degradation, and this might suggest similar nucleophilic substitution at P centers and C-P bond cleavage both in manganese oxide- and UV light-mediated degradation. The DFT results reveal that the C-P bond could be cleaved by water, OH - or • OH, with the energy barrier opposing bond dissociation being lowest in the presence of the radical species, and that C-N bond cleavage is favored by the formation of both nitrogen- and carbon-centered radicals. Overall, these results highlight the factors controlling the dominance of C-P or C-N bond cleavage that determines the composition of intermediate/final products and ultimately the degradation pathway.

  20. Mo(CO)/sub 6/-promoted reductive cleavage of the carbon-sulfur bond

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Luh, T.Y.; Wong, C.S.

    1985-12-13

    In order to study the reductive cleavage of carbon-sulfur bonds by Mo(CO/sub 6/, various organosulfur compounds are reacted with Mo(CO)/sub 6/ in THF. Results of these experiments demonstrate that benzylic-, aryl-, or ..cap alpha..-acyl-activated carbon-sulfur bonds are reduced by treatment with Mo(CO)/sub 6/. 1 table.

  1. Changes in antioxidant capacity and colour associated with the formation of β-carotene epoxides and oxidative cleavage derivatives.

    PubMed

    Gurak, Poliana D; Mercadante, Adriana Z; González-Miret, M L; Heredia, Francisco J; Meléndez-Martínez, Antonio J

    2014-03-15

    In this study HPLC-DAD-MS/MS was applied for the identification of compounds derived from (all-E)-β-carotene following epoxidation and oxidative cleavage. The consequences on the CIELAB colour parameters and antioxidant capacity (AC) were also evaluated. Five apocarotenoids, three secocarotenoids, seven Z isomers and two epoxides were detected as a result of the oxidative cleavage. Four epoxides and three Z isomers were detected as a consequence of the epoxidation reaction. Some compounds were detected for the first time as a result of oxidation reactions. Both treatments led to a marked decrease in b(∗) and Cab(∗) values, indicating that these colour parameters can be used for the rapid assessment of β-carotene oxidation. The oxidative cleavage of β-carotene resulted in increased capacity to both scavenge ABTS(+) and quench singlet oxygen. These results suggest that the study of the AC of these oxidative derivatives and their possible usefulness as food ingredients deserves further attention. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Selmer, B.L.; Hanecak, R.; Anderson, C.W.

    1981-01-01

    Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a singly (virus-encoded.) proteinase. The amino terminus of P2-3b, on the other hand, ismore » produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381-3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein.« less

  3. [Challenges and risks in the development of the ego ideal in adolescence].

    PubMed

    Helbing-Tietze, Brigitte

    2003-11-01

    The author proposes to speak of representations concerning the ideal self, the ideal relationship, the ideal society instead of ego ideal. An active self develops ideals and uses them as standards for orientation, to regulate the affects, and to fulfill needs. The different ideals often do not fit together and are therefore difficult to realize. Adolescents normally refuse their parents' ideals and create new ones with the help of their peers. This developmental step is full of challenges and risks as will be explained in this article.

  4. The ideal subject distance for passport pictures.

    PubMed

    Verhoff, Marcel A; Witzel, Carsten; Kreutz, Kerstin; Ramsthaler, Frank

    2008-07-04

    In an age of global combat against terrorism, the recognition and identification of people on document images is of increasing significance. Experiments and calculations have shown that the camera-to-subject distance - not the focal length of the lens - can have a significant effect on facial proportions. Modern passport pictures should be able to function as a reference image for automatic and manual picture comparisons. This requires a defined subject distance. It is completely unclear which subject distance, in the taking of passport photographs, is ideal for the recognition of the actual person. We show here that the camera-to-subject distance that is perceived as ideal is dependent on the face being photographed, even if the distance of 2m was most frequently preferred. So far the problem of the ideal camera-to-subject distance for faces has only been approached through technical calculations. We have, for the first time, answered this question experimentally with a double-blind experiment. Even if there is apparently no ideal camera-to-subject distance valid for every face, 2m can be proposed as ideal for the taking of passport pictures. The first step would actually be the determination of a camera-to-subject distance for the taking of passport pictures within the standards. From an anthropological point of view it would be interesting to find out which facial features allow the preference of a shorter camera-to-subject distance and which allow the preference of a longer camera-to-subject distance.

  5. Sequence-specific DNA cleavage by Fe2+-mediated fenton reactions has possible biological implications.

    PubMed

    Henle, E S; Han, Z; Tang, N; Rai, P; Luo, Y; Linn, S

    1999-01-08

    Preferential cleavage sites have been determined for Fe2+/H2O2-mediated oxidations of DNA. In 50 mM H2O2, preferential cleavages occurred at the nucleoside 5' to each of the dG moieties in the sequence RGGG, a sequence found in a majority of telomere repeats. Within a plasmid containing a (TTAGGG)81 human telomere insert, 7-fold more strand breakage occurred in the restriction fragment with the insert than in a similar-sized control fragment. This result implies that telomeric DNA could protect coding DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and aging. In micromolar H2O2, preferential cleavage occurred at the thymidine within the sequence RTGR, a sequence frequently found to be required in promoters for normal responses of many procaryotic and eucaryotic genes to iron or oxygen stress. Computer modeling of the interaction of Fe2+ with RTGR in B-DNA suggests that due to steric hindrance with the thymine methyl, Fe2+ associates in a specific manner with the thymine flipped out from the base stack so as to allow an octahedrally-oriented coordination of the Fe2+ with the three purine N7 residues. Fe2+-dependent changes in NMR spectra of duplex oligonucleotides containing ATGA versus those containing AUGA or A5mCGA were consistent with this model.

  6. High-fat diet feeding causes rapid, non-apoptotic cleavage of caspase-3 in astrocytes.

    PubMed

    Guyenet, Stephan J; Nguyen, Hong T; Hwang, Bang H; Schwartz, Michael W; Baskin, Denis G; Thaler, Joshua P

    2013-05-28

    Astrocytes respond to multiple forms of central nervous system (CNS) injury by entering a reactive state characterized by morphological changes and a specific pattern of altered protein expression. Termed astrogliosis, this response has been shown to strongly influence the injury response and functional recovery of CNS tissues. This pattern of CNS inflammation and injury associated with astrogliosis has recently been found to occur in the energy homeostasis centers of the hypothalamus during diet-induced obesity (DIO) in rodent models, but the characterization of the astrocyte response remains incomplete. Here, we report that astrocytes in the mediobasal hypothalamus respond robustly and rapidly to purified high-fat diet (HFD) feeding by cleaving caspase-3, a protease whose cleavage is often associated with apoptosis. Although obesity develops in HFD-fed rats by day 14, caspase-3 cleavage occurs by day 3, prior to the development of obesity, suggesting the possibility that it could play a causal role in the hypothalamic neuropathology and fat gain observed in DIO. Caspase-3 cleavage is not associated with an increase in the rate of apoptosis, as determined by TUNEL staining, suggesting it plays a non-apoptotic role analogous to the response to excitotoxic neuron injury. Our results indicate that astrocytes in the mediobasal hypothalamus respond rapidly and robustly to HFD feeding, activating caspase-3 in the absence of apoptosis, a process that has the potential to influence the course of DIO. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Spaces of ideal convergent sequences.

    PubMed

    Mursaleen, M; Sharma, Sunil K

    2014-01-01

    In the present paper, we introduce some sequence spaces using ideal convergence and Musielak-Orlicz function ℳ = (M(k)). We also examine some topological properties of the resulting sequence spaces.

  8. Effect of solution non-ideality on erythrocyte volume regulation.

    PubMed

    Levin, R L; Cravalho, E G; Huggins, C E

    1977-03-01

    A non-ideal, hydrated, non-dilute pseudo-binary salt-protein-water solution model of the erythrocyte intracellular solution is presented to describe the osmotic behavior of human erythrocytes. Existing experimental activity data for salts and proteins in aqueous solutions are used to formulate van Laar type expressions for the solvent and solute activity coefficients. Reasonable estimates can therefore be made of the non-ideality of the erythrocyte intracellular solution over a wide range of osmolalities. Solution non-ideality is shown to affect significantly the degree of solute polarization within the erythrocyte intracellular solution during freezing. However, the non-ideality has very little effect upon the amount of water retained within erythrocytes cooled at sub-zero temperatures.

  9. Media-portrayed idealized images, body shame, and appearance anxiety.

    PubMed

    Monro, Fiona; Huon, Gail

    2005-07-01

    This study was designed to determine the effects of media-portrayed idealized images on young women's body shame and appearance anxiety, and to establish whether the effects depend on advertisement type and on participant self-objectification. Participants were 39 female university students. Twenty-four magazine advertisements comprised 12 body-related and 12 non-body-related products, one half of each with, and the other one half without, idealized images. Preexposure and post exposure body shame and appearance anxiety measures were recorded. Appearance anxiety increased after viewing advertisements featuring idealized images. There was also a significant interaction between self-objectification level and idealized body (presence vs. absence). No differences emerged for body-related compared with non-body-related product advertisements. The only result for body shame was a main effect for time. Participants' body shame increased after exposure to idealized images, irrespective of advertisement type. Although our findings reveal that media-portrayed idealized images detrimentally affect the body image of young women, they highlight the individual differences in vulnerability and the different effects for different components of body image. These results are discussed in terms of their implications for the prevention and early intervention of body image and dieting-related disorders. ( Copyright 2005 by Wiley Periodicals, Inc

  10. Modeling the MHC class I pathway by combining predictions of proteasomal cleavage, TAP transport and MHC class I binding.

    PubMed

    Tenzer, S; Peters, B; Bulik, S; Schoor, O; Lemmel, C; Schatz, M M; Kloetzel, P-M; Rammensee, H-G; Schild, H; Holzhütter, H-G

    2005-05-01

    Epitopes presented by major histocompatibility complex (MHC) class I molecules are selected by a multi-step process. Here we present the first computational prediction of this process based on in vitro experiments characterizing proteasomal cleavage, transport by the transporter associated with antigen processing (TAP) and MHC class I binding. Our novel prediction method for proteasomal cleavages outperforms existing methods when tested on in vitro cleavage data. The analysis of our predictions for a new dataset consisting of 390 endogenously processed MHC class I ligands from cells with known proteasome composition shows that the immunological advantage of switching from constitutive to immunoproteasomes is mainly to suppress the creation of peptides in the cytosol that TAP cannot transport. Furthermore, we show that proteasomes are unlikely to generate MHC class I ligands with a C-terminal lysine residue, suggesting processing of these ligands by a different protease that may be tripeptidyl-peptidase II (TPPII).

  11. Similarity solutions for unsteady flow behind an exponential shock in a self-gravitating non-ideal gas with azimuthal magnetic field

    NASA Astrophysics Data System (ADS)

    Nath, G.; Pathak, R. P.; Dutta, Mrityunjoy

    2018-01-01

    Similarity solutions for the flow of a non-ideal gas behind a strong exponential shock driven out by a piston (cylindrical or spherical) moving with time according to an exponential law is obtained. Solutions are obtained, in both the cases, when the flow between the shock and the piston is isothermal or adiabatic. The shock wave is driven by a piston moving with time according to an exponential law. Similarity solutions exist only when the surrounding medium is of constant density. The effects of variation of ambient magnetic field, non-idealness of the gas, adiabatic exponent and gravitational parameter are worked out in detail. It is shown that the increase in the non-idealness of the gas or the adiabatic exponent of the gas or presence of magnetic field have decaying effect on the shock wave. Consideration of the isothermal flow and the self-gravitational field increase the shock strength. Also, the consideration of isothermal flow or the presence of magnetic field removes the singularity in the density distribution, which arises in the case of adiabatic flow. The result of our study may be used to interpret measurements carried out by space craft in the solar wind and in neighborhood of the Earth's magnetosphere.

  12. Non-Ideality in Solvent Extraction Systems: PNNL FY 2014 Status Report

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Levitskaia, Tatiana G.; Chatterjee, Sayandev; Pence, Natasha K.

    The overall objective of this project is to develop predictive modeling capabilities for advanced fuel cycle separation processes by gaining a fundamental quantitative understanding of non-ideality effects and speciation in relevant aqueous and organic solutions. Aqueous solutions containing actinides and lanthanides encountered during nuclear fuel reprocessing have high ionic strength and do not behave as ideal solutions. Activity coefficients must be calculated to take into account the deviation from ideality and predict their behavior. In FY 2012-2013, a convenient method for determining activity effects in aqueous electrolyte solutions was developed. Our initial experiments demonstrated that water activity and osmotic coefficientsmore » of the electrolyte solutions can be accurately measured by the combination of two techniques, a Water Activity Meter and Vapor Pressure Osmometry (VPO). The water activity measurements have been conducted for binary lanthanide solutions in wide concentration range for all lanthanides (La-Lu with the exception of Pm). The osmotic coefficients and Pitzer parameters for each binary system were obtained by the least squares fitting of the water activity data. However, application of Pitzer model for the quantitative evaluation of the activity effects in the multicomponent mixtures is difficult due to the large number of the required interaction parameters. In FY 2014, the applicability of the Bromley model for the determination of the Ln(NO 3) 3 activity coefficients was evaluated. The new Bromley parameters for the binary Ln(NO 3) 3 electrolytes were obtained based on the available literature and our experimental data. This allowed for the accurate prediction of the Ln(NO 3) 3 activity coefficients for the binary Ln(NO 3) 3 electrolytes. This model was then successfully implemented for the determination of the Ln(NO 3) 3 activity coefficients in the ternary Nd(NO 3) 3/HNO 3/H2O, Eu(NO 3) 3/HNO 3/H 2O, and Eu(NO 3) 3/NaNO 3/H 2O

  13. Proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis in A431 cells.

    PubMed

    Tang, T K; Chang, W C; Chan, W H; Yang, S D; Ni, M H; Yu, J S

    1998-09-15

    Exposure of mammalian cells to ultraviolet (UV) light elicits a cellular response and can also lead to apoptotic cell death. In this report, we show that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be dramatically activated during the early stages of UV irradiation-triggered apoptosis of A431 cells. Immunoblot analysis revealed that this 36-kDa MBP kinase could be recognized by an antibody against the C-terminal regions of a family of p21Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as studying tools, we further demonstrated that UV irradiation caused cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment and a 30-kDa N-terminal fragment in A431 cells. The appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in A431 cells upon UV irradiation. In addition, UV irradiation also led to activation of CPP32/caspase-3, but not ICH-1L/caspase-2 and ICE/caspase-1, in A431 cells and the kinetics of activation of CPP32/caspase-3 appeared to correlate well with that of DNA fragmentation and of cleavage/activation of PAK2, respectively. Moreover, blockage of activation of CPP32/caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors for caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could significantly attenuate the extent of cleavage/activation of PAK2 induced by UV irradiation. Collectively, the results demonstrate that cleavage and activation of PAK2 can be induced during the early stages of UV irradiation-triggered apoptosis and indicate the involvement of CPP32/caspase-3 in this process.

  14. Characterization of a backbone cleavage product of BMS-196854 (Oncostatin M), a recombinant anti-inflammatory cytokine.

    PubMed

    Zhao, F; Stein, D J; Paborji, M; Cash, P W; Root, B J; Wei, Z; Knupp, C J

    2001-01-01

    BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein. SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies. Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction. Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin

  15. The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo.

    PubMed

    Capmany, G; Taylor, A; Braude, P R; Bolton, V N

    1996-05-01

    The timing of pronuclear formation and breakdown, DNA synthesis and cleavage during the first cell cycle of human embryogenesis are described. Pronuclei formed between 3 and 10 h post-insemination (hpi; median 8 hpi). S-phase commenced between 8 and 14 hpi, and was completed between 10 and 18 hpi. M-phase was observed between 22 and 31 hpi (median duration 3 h), and cleavage to the 2-cell stage took place between 25 and 33 hpi. The timing of the same events was determined in 1-cell embryos derived from re-inseminated human oocytes that had failed to fertilize during therapeutic in-vitro fertilization (IVF). In these embryos, pronuclei formed between 3 and 8 h post-re-insemination (hpr-i), coinciding with the beginning of S-phase. While S-phase was completed as early as 10 hpr-i in some embryos, it extended until at least 16 hpr-i in others. Pronuclear breakdown and cleavage occurred from 23 and 26 hpr-i respectively; however, they did not occur in some embryos until after 46 hpr-i. The results demonstrate a markedly greater degree of variation in the timing of these events in embryos derived from re-inseminated oocytes compared with embryos derived from conventional IVF, and thus throw into question the validity of using the former as models for studies of the first cell cycle of human embryogenesis.

  16. Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage

    PubMed Central

    Jennebach, Stefan; Herzog, Franz; Aebersold, Ruedi; Cramer, Patrick

    2012-01-01

    RNA polymerase (Pol) I contains a 10-subunit catalytic core that is related to the core of Pol II and includes subunit A12.2. In addition, Pol I contains the heterodimeric subcomplexes A14/43 and A49/34.5, which are related to the Pol II subcomplex Rpb4/7 and the Pol II initiation factor TFIIF, respectively. Here we used lysine-lysine crosslinking, mass spectrometry (MS) and modeling based on five crystal structures, to extend the previous homology model of the Pol I core, to confirm the location of A14/43 and to position A12.2 and A49/34.5 on the core. In the resulting model of Pol I, the C-terminal ribbon (C-ribbon) domain of A12.2 reaches the active site via the polymerase pore, like the C-ribbon of the Pol II cleavage factor TFIIS, explaining why the intrinsic RNA cleavage activity of Pol I is strong, in contrast to the weak cleavage activity of Pol II. The A49/34.5 dimerization module resides on the polymerase lobe, like TFIIF, whereas the A49 tWH domain resides above the cleft, resembling parts of TFIIE. This indicates that Pol I and also Pol III are distantly related to a Pol II–TFIIS–TFIIF–TFIIE complex. PMID:22396529

  17. Coupling Ideality of Integrated Planar High-Q Microresonators

    NASA Astrophysics Data System (ADS)

    Pfeiffer, Martin H. P.; Liu, Junqiu; Geiselmann, Michael; Kippenberg, Tobias J.

    2017-02-01

    Chip-scale optical microresonators with integrated planar optical waveguides are useful building blocks for linear, nonlinear, and quantum-optical photonic devices alike. Loss reduction through improving fabrication processes results in several integrated microresonator platforms attaining quality (Q ) factors of several millions. Beyond the improvement of the quality factor, the ability to operate the microresonator with high coupling ideality in the overcoupled regime is of central importance. In this regime, the dominant source of loss constitutes the coupling to a single desired output channel, which is particularly important not only for quantum-optical applications such as the generation of squeezed light and correlated photon pairs but also for linear and nonlinear photonics. However, to date, the coupling ideality in integrated photonic microresonators is not well understood, in particular, design-dependent losses and their impact on the regime of high ideality. Here we investigate design-dependent parasitic losses described by the coupling ideality of the commonly employed microresonator design consisting of a microring-resonator waveguide side coupled to a straight bus waveguide, a system which is not properly described by the conventional input-output theory of open systems due to the presence of higher-order modes. By systematic characterization of multimode high-Q silicon nitride microresonator devices, we show that this design can suffer from low coupling ideality. By performing 3D simulations, we identify the coupling to higher-order bus waveguide modes as the dominant origin of parasitic losses which lead to the low coupling ideality. Using suitably designed bus waveguides, parasitic losses are mitigated with a nearly unity ideality and strong overcoupling (i.e., a ratio of external coupling to internal resonator loss rate >9 ) are demonstrated. Moreover, we find that different resonator modes can exchange power through the coupler, which, therefore

  18. Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes.

    PubMed

    Bonilla, Julia B; Cid, M Belén; Contreras, F-Xabier; Goñi, Félix M; Martín-Lomas, Manuel

    2006-02-01

    The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs.

  19. Structural basis for activation of the complement system by component C4 cleavage

    PubMed Central

    Kidmose, Rune T.; Laursen, Nick S.; Dobó, József; Kjaer, Troels R.; Sirotkina, Sofia; Yatime, Laure; Sottrup-Jensen, Lars; Thiel, Steffen; Gál, Péter; Andersen, Gregers R.

    2012-01-01

    An essential aspect of innate immunity is recognition of molecular patterns on the surface of pathogens or altered self through the lectin and classical pathways, two of the three well-established activation pathways of the complement system. This recognition causes activation of the MASP-2 or the C1s serine proteases followed by cleavage of the protein C4. Here we present the crystal structures of the 203-kDa human C4 and the 245-kDa C4⋅MASP-2 substrate⋅enzyme complex. When C4 binds to MASP-2, substantial conformational changes in C4 are induced, and its scissile bond region becomes ordered and inserted into the protease catalytic site in a manner canonical to serine proteases. In MASP-2, an exosite located within the CCP domains recognizes the C4 C345C domain 60 Å from the scissile bond. Mutations in C4 and MASP-2 residues at the C345C–CCP interface inhibit the intermolecular interaction and C4 cleavage. The possible assembly of the huge in vivo enzyme–substrate complex consisting of glycan-bound mannan-binding lectin, MASP-2, and C4 is discussed. Our own and prior functional data suggest that C1s in the classical pathway of complement activated by, e.g., antigen–antibody complexes, also recognizes the C4 C345C domain through a CCP exosite. Our results provide a unified structural framework for understanding the early and essential step of C4 cleavage in the elimination of pathogens and altered self through two major pathways of complement activation. PMID:22949645

  20. Strength, hardness, and lattice vibrations of Z-carbon and W-carbon: First-principles calculations

    NASA Astrophysics Data System (ADS)

    Li, Zhiping; Gao, Faming; Xu, Ziming

    2012-04-01

    The strength, hardness, and lattice vibrations of two superhard carbon allotropies, Z-carbon and W-carbon are investigated by first-principles calculations. Phonon dispersion calculations indicate that Z-carbon and W-carbon are dynamically stable at least up to 300 GPa. The strength calculations reveal that the failure mode in Z-carbon is dominated by the tensile type, and the [010] direction is the weakest one. In W-carbon, the failure mode is dominated by the shear type, and the (101)[111¯] direction is the weakest one. Although the ideal strength of diamond is distinctly greater than that of Z-carbon and W-carbon, the tensile strength and shear strength for Z-carbon and W-carbon show much lower anisotropies than that of diamond. The hardness calculations indicate that the average hardness of Z-carbon is less than that of diamond but greater than that of the W-carbon, M-carbon, and body-centered-tetragonal-C4 carbon. The simulated Raman spectra show that the Ag modes at 1094 cm-1 for Z-carbon and 1109.7 cm-1 for W-carbon are in agreement with that of 1082 cm-1 observed in the experiment of cold-compressed graphite at 9.8 GPa.

  1. Mitochondrial dysfunction-associated OPA1 cleavage contributes to muscle degeneration: preventative effect of hydroxytyrosol acetate.

    PubMed

    Wang, X; Li, H; Zheng, A; Yang, L; Liu, J; Chen, C; Tang, Y; Zou, X; Li, Y; Long, J; Liu, J; Zhang, Y; Feng, Z

    2014-11-13

    Mitochondrial dysfunction contributes to the development of muscle disorders, including muscle wasting, muscle atrophy and degeneration. Despite the knowledge that oxidative stress closely interacts with mitochondrial dysfunction, the detailed mechanisms remain obscure. In this study, tert-butylhydroperoxide (t-BHP) was used to induce oxidative stress on differentiated C2C12 myotubes. t-BHP induced significant mitochondrial dysfunction in a time-dependent manner, accompanied by decreased myosin heavy chain (MyHC) expression at both the mRNA and protein levels. Consistently, endogenous reactive oxygen species (ROS) overproduction triggered by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a mitochondrial oxidative phosphorylation inhibitor, was accompanied by decreased membrane potential and decreased MyHC protein content. However, the free radical scavenger N-acetyl-L-cysteine (NAC) efficiently reduced the ROS level and restored MyHC content, suggesting a close association between ROS and MyHC expression. Meanwhile, we found that both t-BHP and FCCP promoted the cleavage of optic atrophy 1 (OPA1) from the long form into short form during the early stages. In addition, the ATPase family gene 3-like 2, a mitochondrial inner membrane protease, was also markedly increased. Moreover, OPA1 knockdown in myotubes was accompanied by decreased MyHC content, whereas NAC failed to prevent FCCP-induced MyHC decrease with OPA1 knockdown, suggesting that ROS might affect MyHC content by modulating OPA1 cleavage. In addition, hydroxytyrosol acetate (HT-AC), an important compound in virgin olive oil, could significantly prevent t-BHP-induced mitochondrial membrane potential and cell viability loss in myotubes. Specifically, HT-AC inhibited t-BHP-induced OPA1 cleavage and mitochondrial morphology changes, accompanied by improvement on mitochondrial oxygen consumption capacity, ATP productive potential and activities of mitochondrial complex I, II and V. Moreover, both

  2. Non-ideal magnetohydrodynamics on a moving mesh

    NASA Astrophysics Data System (ADS)

    Marinacci, Federico; Vogelsberger, Mark; Kannan, Rahul; Mocz, Philip; Pakmor, Rüdiger; Springel, Volker

    2018-05-01

    In certain astrophysical systems, the commonly employed ideal magnetohydrodynamics (MHD) approximation breaks down. Here, we introduce novel explicit and implicit numerical schemes of ohmic resistivity terms in the moving-mesh code AREPO. We include these non-ideal terms for two MHD techniques: the Powell 8-wave formalism and a constrained transport scheme, which evolves the cell-centred magnetic vector potential. We test our implementation against problems of increasing complexity, such as one- and two-dimensional diffusion problems, and the evolution of progressive and stationary Alfvén waves. On these test problems, our implementation recovers the analytic solutions to second-order accuracy. As first applications, we investigate the tearing instability in magnetized plasmas and the gravitational collapse of a rotating magnetized gas cloud. In both systems, resistivity plays a key role. In the former case, it allows for the development of the tearing instability through reconnection of the magnetic field lines. In the latter, the adopted (constant) value of ohmic resistivity has an impact on both the gas distribution around the emerging protostar and the mass loading of magnetically driven outflows. Our new non-ideal MHD implementation opens up the possibility to study magneto-hydrodynamical systems on a moving mesh beyond the ideal MHD approximation.

  3. Cleavage of cohesin rings coordinates the separation of centrioles and chromatids.

    PubMed

    Schöckel, Laura; Möckel, Martin; Mayer, Bernd; Boos, Dominik; Stemmann, Olaf

    2011-07-10

    Cohesin pairs sister chromatids by forming a tripartite Scc1-Smc1-Smc3 ring around them. In mitosis, cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4-6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 is replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex.

  4. Tibiofemoral contact mechanics following a horizontal cleavage lesion in the posterior horn of the medial meniscus.

    PubMed

    Arno, Sally; Bell, Christopher P; Uquillas, Carlos; Borukhov, Ilya; Walker, Peter S

    2015-04-01

    The purpose of this study was to determine if a horizontal cleavage lesion (HCL) of the posterior horn of the medial meniscus would result in changes to tibiofemoral contact mechanics, as measured by peak contact pressure and contact area, which can lead to cartilage degeneration. To study this, 10 cadaveric knees were tested in a rig where forces were applied (500 N Compression, 100 N shear, 2.5 Nm Torque) and the knee dynamically flexed from -5° to 135°, as peak contact pressure and contact area were recorded. After testing of the intact knee, a horizontal cleavage lesion was created arthroscopically and testing repeated. The Wilcoxon signed-rank test was used to determine if there were differences in peak contact pressure and contact area between the intact knee and that with the HCL. A statistically significant increase in peak contact pressure of 13%, on average, and a decrease in contact area of 6%, on average, was noted following the HCL. This suggests that a horizontal cleavage lesion will result in small but statistically significant changes in tibiofemoral contact mechanics which may lead to cartilage degeneration. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  5. Distinct oxidative cleavage and modification of bovine [Cu-Zn]-SOD by an ascorbic acid/Cu(II) system: Identification of novel copper binding site on SOD molecule

    PubMed Central

    Uehara, Hiroshi; Luo, Shen; Aryal, Baikuntha; Levine, Rodney L.; Rao, V. Ashutosh

    2016-01-01

    We investigated the combined effect of ascorbate and copper [Asc/Cu(II)] on the integrity of bovine [Cu-Zn]-superoxide dismutase (bSOD1) as a model system to study the metal catalyzed oxidation (MCO) and fragmentation of proteins. We found Asc/Cu(II) mediates specific cleavage of bSOD1 and generates 12.5 and 3.2 kDa fragments in addition to oxidation/carbonylation of the protein. The effect of other tested transition metals, a metal chelator, and hydrogen peroxide on the cleavage and oxidation indicated that binding of copper to a previously unknown site on SOD1 is responsible for the Asc/Cu(II) specific cleavage and oxidation. We utilized tandem mass spectrometry to identify the specific cleavage sites of Asc/Cu(II)-treated bSOD1. Analyses of tryptic- and AspN-peptides have demonstrated the cleavage to occur at Gly31 with peptide bond breakage with Thr30 and Ser32 through diamide and α-amidation pathways, respectively. The three-dimensional structure of bSOD1 reveals the imidazole ring of His19 localized within 5 Angstrom from the α-carbon of Gly31 providing a structural basis that copper ion, most likely coordinated by His19, catalyzes the specific cleavage reaction. PMID:26872685

  6. Distinct oxidative cleavage and modification of bovine [Cu- Zn]-SOD by an ascorbic acid/Cu(II) system: Identification of novel copper binding site on SOD molecule.

    PubMed

    Uehara, Hiroshi; Luo, Shen; Aryal, Baikuntha; Levine, Rodney L; Rao, V Ashutosh

    2016-05-01

    We investigated the combined effect of ascorbate and copper [Asc/Cu(II)] on the integrity of bovine [Cu-Zn]-superoxide dismutase (bSOD1) as a model system to study the metal catalyzed oxidation (MCO) and fragmentation of proteins. We found Asc/Cu(II) mediates specific cleavage of bSOD1 and generates 12.5 and 3.2kDa fragments in addition to oxidation/carbonylation of the protein. The effect of other tested transition metals, a metal chelator, and hydrogen peroxide on the cleavage and oxidation indicated that binding of copper to a previously unknown site on SOD1 is responsible for the Asc/Cu(II) specific cleavage and oxidation. We utilized tandem mass spectrometry to identify the specific cleavage sites of Asc/Cu(II)-treated bSOD1. Analyses of tryptic- and AspN-peptides have demonstrated the cleavage to occur at Gly31 with peptide bond breakage with Thr30 and Ser32 through diamide and α-amidation pathways, respectively. The three-dimensional structure of bSOD1 reveals the imidazole ring of His19 localized within 5Å from the α-carbon of Gly31 providing a structural basis that copper ion, most likely coordinated by His19, catalyzes the specific cleavage reaction. Published by Elsevier Inc.

  7. Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage.

    PubMed

    Dayeh, Daniel M; Kruithoff, Bradley C; Nakanishi, Kotaro

    2018-04-27

    Some gene transcripts have cellular functions as regulatory noncoding RNAs. For example, ∼23-nucleotide (nt)-long siRNAs are loaded into Argonaute proteins. The resultant ribonucleoprotein assembly, the RNA-induced silencing complex (RISC), cleaves RNAs that are extensively base-paired with the loaded siRNA. To date, base complementarity is recognized as the major determinant of specific target cleavage (or slicing), but little is known about how Argonaute inspects base pairing before cleavage. A hallmark of Argonaute proteins is their bilobal structure, but despite the significance of this structure for curtailing slicing activity against mismatched targets, the molecular mechanism remains elusive. Here, our structural and functional studies of a bilobed yeast Argonaute protein and its isolated catalytic C-terminal lobe (C-lobe) revealed that the C-lobe alone retains almost all properties of bilobed Argonaute: siRNA-duplex loading, passenger cleavage/ejection, and siRNA-dependent RNA cleavage. A 2.1 Å-resolution crystal structure revealed that the catalytic C-lobe mirrors the bilobed Argonaute in terms of guide-RNA recognition and that all requirements for transitioning to the catalytically active conformation reside in the C-lobe. Nevertheless, we found that in the absence of the N-terminal lobe (N-lobe), target RNAs are scanned for complementarity only at positions 5-14 on a 23-nt guide RNA before endonucleolytic cleavage, thereby allowing for some off-target cleavage. Of note, acquisition of an N-lobe expanded the range of the guide RNA strand used for inspecting target complementarity to positions 2-23. These findings offer clues to the evolution of the bilobal structure of catalytically active Argonaute proteins. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Chain Ends and the Ultimate Tensile Strength of Polyethylene Fibers

    NASA Astrophysics Data System (ADS)

    O'Connor, Thomas C.; Robbins, Mark O.

    Determining the tensile yield mechanisms of oriented polymer fibers remains a challenging problem in polymer mechanics. By maximizing the alignment and crystallinity of polyethylene (PE) fibers, tensile strengths σ ~ 6 - 7 GPa have been achieved. While impressive, first-principal calculations predict carbon backbone bonds would allow strengths four times higher (σ ~ 20 GPa) before breaking. The reduction in strength is caused by crystal defects like chain ends, which allow fibers to yield by chain slip in addition to bond breaking. We use large scale molecular dynamics (MD) simulations to determine the tensile yield mechanism of orthorhombic PE crystals with finite chains spanning 102 -104 carbons in length. The yield stress σy saturates for long chains at ~ 6 . 3 GPa, agreeing well with experiments. Chains do not break but always yield by slip, after nucleation of 1D dislocations at chain ends. Dislocations are accurately described by a Frenkel-Kontorova model, parametrized by the mechanical properties of an ideal crystal. We compute a dislocation core size ξ = 25 . 24 Å and determine the high and low strain rate limits of σy. Our results suggest characterizing such 1D dislocations is an efficient method for predicting fiber strength. This research was performed within the Center for Materials in Extreme Dynamic Environments (CMEDE) under the Hopkins Extreme Materials Institute at Johns Hopkins University. Financial support was provided by Grant W911NF-12-2-0022.

  9. Kallistatin Ameliorates Influenza Virus Pathogenesis by Inhibition of Kallikrein-Related Peptidase 1-Mediated Cleavage of Viral Hemagglutinin

    PubMed Central

    Leu, Chia-Hsing; Yang, Mei-Lin; Chung, Nai-Hui; Huang, Yen-Jang; Su, Yu-Chu; Chen, Yi-Cheng; Lin, Chia-Cheng; Shieh, Gia-Shing; Chang, Meng-Ya; Wang, Shainn-Wei; Chang, Yao; Chao, Julie; Chao, Lee

    2015-01-01

    Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses. PMID:26149981

  10. Molecular pathogenesis of H5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif

    PubMed Central

    Luczo, Jasmina M.; Stambas, John; Durr, Peter A.; Michalski, Wojtek P.

    2015-01-01

    Summary The emergence of H5N1 highly pathogenic avian influenza has caused a heavy socio‐economic burden through culling of poultry to minimise human and livestock infection. Although human infections with H5N1 have to date been limited, concerns for the pandemic potential of this zoonotic virus have been greatly intensified following experimental evidence of aerosol transmission of H5N1 viruses in a mammalian infection model. In this review, we discuss the dominance of the haemagglutinin cleavage site motif as a pathogenicity determinant, the host‐pathogen molecular interactions driving cleavage activation, reverse genetics manipulations and identification of residues key to haemagglutinin cleavage site functionality and the mechanisms of cell and tissue damage during H5N1 infection. We specifically focus on the disease in chickens, as it is in this species that high pathogenicity frequently evolves and from which transmission to the human population occurs. With >75% of emerging infectious diseases being of zoonotic origin, it is necessary to understand pathogenesis in the primary host to explain spillover events into the human population. © 2015 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd. PMID:26467906

  11. Sharp Truncation of an Electric Field: An Idealized Model that Warrants Caution

    NASA Astrophysics Data System (ADS)

    Tu, Hong; Zhu, Jiongming

    2016-03-01

    In physics, idealized models are often used to simplify complex situations. The motivation of the idealization is to make the real complex system tractable by adopting certain simplifications. In this treatment some unnecessary, negligible aspects are stripped away (so-called Aristotelian idealization), or some deliberate distortions are involved (so-called Galilean idealization). The most important principle in using an idealized model is to make sure that all the neglected aspects do not affect our analysis or result. Point charges, rigid bodies, simple pendulums, frictionless planes, and isolated systems are all frequently used idealized models. However, when they are applied to certain uncommon models, extra precautions should be taken. The possibilities and necessities of adopting the idealizations have to be considered carefully. Sometimes some factors neglected or ignored in the idealization could completely change the result, even make the treatment unphysical and conclusions unscientific.

  12. Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons.

    PubMed

    Hendrickson, Peter G; Doráis, Jessie A; Grow, Edward J; Whiddon, Jennifer L; Lim, Jong-Won; Wike, Candice L; Weaver, Bradley D; Pflueger, Christian; Emery, Benjamin R; Wilcox, Aaron L; Nix, David A; Peterson, C Matthew; Tapscott, Stephen J; Carrell, Douglas T; Cairns, Bradley R

    2017-06-01

    To better understand transcriptional regulation during human oogenesis and preimplantation development, we defined stage-specific transcription, which highlighted the cleavage stage as being highly distinctive. Here, we present multiple lines of evidence that a eutherian-specific multicopy retrogene, DUX4, encodes a transcription factor that activates hundreds of endogenous genes (for example, ZSCAN4, KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably, mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells, measured here by the reactivation of '2C' genes and repeat elements, the loss of POU5F1 (also known as OCT4) protein and chromocenters, and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus, we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state.

  13. Alloy and composition dependence of hydrogen embrittlement susceptibility in high-strength steel fasteners

    NASA Astrophysics Data System (ADS)

    Brahimi, S. V.; Yue, S.; Sriraman, K. R.

    2017-06-01

    High-strength steel fasteners characterized by tensile strengths above 1100 MPa are often used in critical applications where a failure can have catastrophic consequences. Preventing hydrogen embrittlement (HE) failure is a fundamental concern implicating the entire fastener supply chain. Research is typically conducted under idealized conditions that cannot be translated into know-how prescribed in fastener industry standards and practices. Additionally, inconsistencies and even contradictions in fastener industry standards have led to much confusion and many preventable or misdiagnosed fastener failures. HE susceptibility is a function of the material condition, which is comprehensively described by the metallurgical and mechanical properties. Material strength has a first-order effect on HE susceptibility, which increases significantly above 1200 MPa and is characterized by a ductile-brittle transition. For a given concentration of hydrogen and at equal strength, the critical strength above which the ductile-brittle transition begins can vary due to second-order effects of chemistry, tempering temperature and sub-microstructure. Additionally, non-homogeneity of the metallurgical structure resulting from poorly controlled heat treatment, impurities and non-metallic inclusions can increase HE susceptibility of steel in ways that are measurable but unpredictable. Below 1200 MPa, non-conforming quality is often the root cause of real-life failures. This article is part of the themed issue 'The challenges of hydrogen and metals'.

  14. Ideal thermodynamic processes of oscillatory-flow regenerative engines will go to ideal stirling cycle?

    NASA Astrophysics Data System (ADS)

    Luo, Ercang

    2012-06-01

    This paper analyzes the thermodynamic cycle of oscillating-flow regenerative machines. Unlike the classical analysis of thermodynamic textbooks, the assumptions for pistons' movement limitations are not needed and only ideal flowing and heat transfer should be maintained in our present analysis. Under such simple assumptions, the meso-scale thermodynamic cycles of each gas parcel in typical locations of a regenerator are analyzed. It is observed that the gas parcels in the regenerator undergo Lorentz cycle in different temperature levels, whereas the locus of all gas parcels inside the regenerator is the Ericson-like thermodynamic cycle. Based on this new finding, the author argued that ideal oscillating-flow machines without heat transfer and flowing losses is not the Stirling cycle. However, this new thermodynamic cycle can still achieve the same efficiency of the Carnot heat engine and can be considered a new reversible thermodynamic cycle under two constant-temperature heat sinks.

  15. Unusual enzymatic glycoside cleavage mechanisms.

    PubMed

    Jongkees, Seino A K; Withers, Stephen G

    2014-01-21

    Over the sixty years since Koshland initially formulated the classical mechanisms for retaining and inverting glycosidases, researchers have assembled a large body of supporting evidence and have documented variations of these mechanisms. Recently, however, researchers have uncovered a number of completely distinct mechanisms for enzymatic cleavage of glycosides involving elimination and/or hydration steps. In family GH4 and GH109 glycosidases, the reaction proceeds via transient NAD(+)-mediated oxidation at C3, thereby acidifying the proton at C2 and allowing for elimination across the C1-C2 bond. Subsequent Michael-type addition of water followed by reduction at C3 generates the hydrolyzed product. Enzymes employing this mechanism can hydrolyze thioglycosides as well as both anomers of activated substrates. Sialidases employ a conventional retaining mechanism in which a tyrosine functions as the nucleophile, but in some cases researchers have observed off-path elimination end products. These reactions occur via the normal covalent intermediate, but instead of an attack by water on the anomeric center, the catalytic acid/base residue abstracts an adjacent proton. These enzymes can also catalyze hydration of the enol ether via the reverse pathway. Reactions of α-(1,4)-glucan lyases also proceed through a covalent intermediate with subsequent abstraction of an adjacent proton to give elimination. However, in this case, the departing carboxylate "nucleophile" serves as the base in a concerted but asynchronous syn-elimination process. These enzymes perform only elimination reactions. Polysaccharide lyases, which act on uronic acid-containing substrates, also catalyze only elimination reactions. Substrate binding neutralizes the charge on the carboxylate, which allows for abstraction of the proton on C5 and leads to an elimination reaction via an E1cb mechanism. These enzymes can also cleave thioglycosides, albeit slowly. The unsaturated product of polysaccharide

  16. Moral Identity as Moral Ideal Self: Links to Adolescent Outcomes

    ERIC Educational Resources Information Center

    Hardy, Sam A.; Walker, Lawrence J.; Olsen, Joseph A.; Woodbury, Ryan D.; Hickman, Jacob R.

    2014-01-01

    The purposes of this study were to conceptualize moral identity as moral ideal self, to develop a measure of this construct, to test for age and gender differences, to examine links between moral ideal self and adolescent outcomes, and to assess purpose and social responsibility as mediators of the relations between moral ideal self and outcomes.…

  17. Racial Cleavage in Local Voting: The Case of School and Tax Issue Referendums.

    ERIC Educational Resources Information Center

    Button, James

    1993-01-01

    Explores voting behavior of African Americans and whites in local school and tax referenda to determine whether racial conflict is still a primal factor in noncandidate elections. Results for voters in 5 counties in Florida (over 1,699,000 voters) reveal African-American underregistration and the continuing importance of racial cleavage. (SLD)

  18. Extending the cleavage rules for the hammerhead ribozyme: mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17.

    PubMed Central

    Ludwig, J; Blaschke, M; Sproat, B S

    1998-01-01

    In this paper, we show that an adenosine to inosine mutation at position 15.1 changes the substrate specificity of the hammerhead ribozyme from N16.2U16.1H17to N16.2C16.1H17(H represents A, C or U). This result extends the hammerhead cleavage triplet definition from N16.2U16.1H17to the more general N16.2Y16.1H17. Comparison of cleavage rates using I15.1ribozymes for NCH triplets and standard A15.1 ribozymes for NUH triplets under single turnover conditions shows similar or slightly enhanced levels of reactivity for the I15. 1-containing structures. The effect of I15.1 substitution was also tested in nuclease-resistant 2'- O -alkyl substituted derivatives (oligozymes), showing a similar level of activity for the NUH and NCH cleaving structures. The availability of NCH triplets that can be targeted without loss of efficiency increases the flexibility of ribozyme targeting strategies. This was demonstrated by an efficient cleavage of an HCV transcript at a previously inaccessible GCA site in codon 2. PMID:9580675

  19. Degradation of Phenolic Compounds and Ring Cleavage of Catechol by Phanerochaete chrysosporium

    PubMed Central

    Leatham, Gary F.; Crawford, R. L.; Kirk, T. Kent

    1983-01-01

    POL-88, a mutant of the white-rot fungus Phanerochaete chrysosporium, was selected for diminished phenol-oxidizing enzyme activity. A wide variety of phenolic compounds were degraded by ligninolytic cultures of this mutant. With several o-diphenolic substrates, degradation intermediates were produced that had UV spectra consistent with muconic acids. Extensive spectrophotometric and polarographic assays failed to detect classical ring-cleaving dioxygenases in cell homogenates or in extracts from ligninolytic cultures. Even so, a sensitive carrier-trapping assay showed that intact cultures degraded [U-14C]catechol to [14C]muconic acid, establishing the presence of a system capable of 1,2-intradiol fission. Significant accumulation of [14C]muconic acid into carrier occurred only when evolution of 14CO2 from [14C]catechol was inhibited by treating cultures with excess nutrient nitrogen (e.g., l-glutamic acid) or with cycloheximide. l-Glutamic acid is known from past work to repress the ligninolytic system in P. chrysosporium and to mimic the effect of cycloheximide. The results here indicate, therefore, that the enzyme system responsible for degrading ring-cleavage products to CO2 turns over faster than does the system responsible for ring cleavage. PMID:16346340

  20. Catalytic Arylation and Vinylation Reactions Directed by Anionic Oxygen Functions via Cleavage of C - H and C - C Bonds

    NASA Astrophysics Data System (ADS)

    Satoh, Tetsuya; Miura, Masahiro

    Aromatic compounds having oxygen-containing substituents such as phenols, phenyl ketones, benzyl alcohols, and benzoic acids undergo regioselective arylation and vinylation via C-H bond cleavage in the presence of transition-metal catalysts. The latter two substrates are also arylated and vinylated via C-C bond cleavage accompanied by liberation of ketones and CO2, respectively. Coordination of their anionic oxygen to the metal center is the key to activate the inert bonds effectively and regioselectively. The recent progress of these oxygen-directed reactions is summarized herein.

  1. The identification of disulfides in ricin D using proteolytic cleavage followed by negative-ion nano-electrospray ionization mass spectrometry of the peptide fragments.

    PubMed

    Tran, T T Nha; Brinkworth, Craig S; Bowie, John H

    2015-01-30

    To use negative-ion nano-electrospray ionization mass spectrometry of peptides from the tryptic digest of ricin D, to provide sequence information; in particular, to identify disulfide position and connectivity. Negative-ion fragmentations of peptides from the tryptic digest of ricin D was studied using a Waters QTOF2 mass spectrometer operating in MS and MS(2) modes. Twenty-three peptides were obtained following high-performance liquid chromatography and studied by negative-ion mass spectrometry covering 73% of the amino-acid residues of ricin D. Five disulfide-containing peptides were identified, three intermolecular and two intramolecular disulfide-containing peptides. The [M-H](-) anions of the intermolecular disulfides undergo facile cleavage of the disulfide units to produce fragment peptides. In negative-ion collision-induced dissociation (CID) these source-formed anions undergo backbone cleavages, which provide sequencing information. The two intramolecular disulfides were converted proteolytically into intermolecular disulfides, which were identified as outlined above. The positions of the five disulfide groups in ricin D may be determined by characteristic negative-ion cleavage of the disulfide groups, while sequence information may be determined using the standard negative-ion backbone cleavages of the resulting cleaved peptides. Negative-ion mass spectrometry can also be used to provide partial sequencing information for other peptides (i.e. those not containing Cys) using the standard negative-ion backbone cleavages of these peptides. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Jung's Red Book and its relation to aspects of German idealism.

    PubMed

    Bishop, Paul

    2012-06-01

    The late nineteenth century saw a renaissance of interest in the thought of the German Romantic philosopher, F.W.J. Schelling. This paper takes Jung's engagement with Schelling and his awareness of Schellingian ideas and interests (notably, the mysterious Kabeiroi worshipped at Samothrace) as its starting-point. It goes on to argue that a key set of problematics in German Idealism - the relation between freedom and necessity, between science and art, and ultimately between realism and idealism - offers a useful conceptual framework within which to approach Jung's Red Book. For the problem of the ideal is central to this work, which can be read as a journey from eternal ideals to the ideal of eternity. (Although the term 'idealism' has at least four distinct meanings, their distinct senses can be related in different ways to Jung's thinking.) The eloquent embrace of idealism by F.T. Vischer in a novel, Auch Einer, for which Jung had the highest praise, reminds us of the persistence of this tradition, which is still contested and debated in the present day. © 2012, The Society of Analytical Psychology.

  3. A Generalized Deduction of the Ideal-Solution Model

    ERIC Educational Resources Information Center

    Leo, Teresa J.; Perez-del-Notario, Pedro; Raso, Miguel A.

    2006-01-01

    A new general procedure for deriving the Gibbs energy of mixing is developed through general thermodynamic considerations, and the ideal-solution model is obtained as a special particular case of the general one. The deduction of the Gibbs energy of mixing for the ideal-solution model is a rational one and viewed suitable for advanced students who…

  4. Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

    PubMed Central

    Henderson, L E; Sowder, R; Copeland, T D; Smythers, G; Oroszlan, S

    1984-01-01

    The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag

  5. Diastereoselective DNA Cleavage Recognition by Ni(II)•Gly-Gly-His Derived Metallopeptides

    PubMed Central

    Fang, Ya-Yin; Claussen, Craig A.; Lipkowitz, Kenny B.; Long, Eric C.

    2008-01-01

    Site-selective DNA cleavage by diastereoisomers of Ni(II)•Gly-Gly-His-derived metallopeptides was investigated through high-resolution gel analyses and molecular dynamics simulations. Ni(II)•L-Arg-Gly-His and Ni(II)•D-Arg-Gly-His (and their respective Lys analogues) targeted A/T-rich regions; however, the L-isomers consistently modified a sub-set of available nucleotides within a given minor groove site while the D-isomers differed in both their sites of preference and ability to target individual nucleotides within some sites. In comparison, Ni(II)•L-Pro-Gly-His and Ni(II)•D-Pro-Gly-His were unable to exhibit a similar diastereoselectivity. Simulations of the above systems, along with Ni(II)•Gly-Gly-His, indicated that the stereochemistry of the amino-terminal amino acid produces either an isohelical metallopeptide that associates stably at individual DNA sites (L-Arg or L-Lys) or, with D-Arg and D-Lys, a non-complementary metallopeptide structure that cannot fully employ its side chain nor amino-terminal amine as a positional stabilizing moiety. In contrast, amino-terminal Pro-containing metallopeptides of either stereochemistry, lacking an extended side chain directed toward the minor groove, did not exhibit a similar diastereoselectivity. While the identity and stereochemistry of amino acids located in the amino-terminal peptide position influenced DNA cleavage, metallopeptide diastereoisomers containing L- and D-Arg (or Lys) within the second peptide position did not exhibit diastereoselective DNA cleavage patterns; simulations indicated that a positively-charged amino acid in this location alters the interaction of the metallopeptide equatorial plane and the minor groove leading to an interaction similar to Ni(II)•Gly-Gly-His. PMID:16522100

  6. Cleavage of an amide bond by a ribozyme

    NASA Technical Reports Server (NTRS)

    Dai, X.; De Mesmaeker, A.; Joyce, G. F.; Miller, S. L. (Principal Investigator)

    1995-01-01

    A variant form of a group I ribozyme, optimized by in vitro evolution for its ability to catalyze magnesium-dependent phosphoester transfer reactions involving DNA substrates, also catalyzes the cleavage of an unactivated alkyl amide when that linkage is presented in the context of an oligodeoxynucleotide analog. Substrates containing an amide bond that joins either two DNA oligos, or a DNA oligo and a short peptide, are cleaved in a magnesium-dependent fashion to generate the expected products. The first-order rate constant, kcat, is 0.1 x 10(-5) min-1 to 1 x 10(-5) min-1 for the DNA-flanked substrates, which corresponds to a rate acceleration of more than 10(3) as compared with the uncatalyzed reaction.

  7. Media-portrayed idealized images, self-objectification, and eating behavior.

    PubMed

    Monro, Fiona J; Huon, Gail F

    2006-11-01

    This study examined the effects of media-portrayed idealized images on young women's eating behavior. The study compared the effects for high and low self-objectifiers. 72 female university students participated in this experiment. Six magazine advertisements featuring idealized female models were used as the experimental stimuli, and the same six advertisements with the idealized body digitally removed became the control stimuli. Eating behavior was examined using a classic taste test that involved both sweet and savory food. Participants' restraint status was assessed. We found that total food intake after exposure was the same in the body present and absent conditions. There were also no differences between high and low self-objectifiers' total food intake. However, for the total amount of food consumed and for sweet food there were significant group by condition interaction effects. High self-objectifiers ate more food in the body present than the body absent condition. In contrast, low self-objectifiers ate more food in the body absent than in the body present condition. Restraint status was not found to moderate the relationship between exposure to idealized images the amount of food consumed. Our results indicate that exposure to media-portrayed idealized images can lead to changes in eating behavior and highlight the complexity of the association between idealized image exposure and eating behavior. These results are discussed in terms of their implications for the prevention of dieting-related disorders.

  8. Polycystin-1 Surface Localization Is Stimulated by Polycystin-2 and Cleavage at the G Protein-coupled Receptor Proteolytic Site

    PubMed Central

    Chapin, Hannah C.; Rajendran, Vanathy

    2010-01-01

    Polycystin (PC)1 and PC2 are membrane proteins implicated in autosomal dominant polycystic kidney disease. A physiologically relevant cleavage at PC1's G protein-coupled receptor proteolytic site (GPS) occurs early in the secretory pathway. Our results suggest that PC2 increases both PC1 GPS cleavage and PC1's appearance at the plasma membrane. Mutations that prevent PC1's GPS cleavage prevent its plasma membrane localization. PC2 is a member of the trp family of cation channels and is an important PC1 binding partner. The effect of PC2 on PC1 localization is independent of PC2 channel activity, as tested using channel-inhibiting PC2 mutations. PC1 and PC2 can interact through their C-terminal tails, but removing the C-terminal tail of either protein has no effect on PC1 surface localization in human embryonic kidney 293 cells. Experiments in polarized LLC-PK cells show that apical and ciliary PC1 localization requires PC2 and that this delivery is sensitive to PC2 truncation. In sum, our work shows that PC2 expression is required for the movement of PC1 to the plasma and ciliary membranes. In fibroblast cells this localization effect is independent of PC2's channel activity or PC1 binding ability but involves a stimulation of PC1's GPS cleavage before the PC1 protein's surface delivery. PMID:20980620

  9. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3

    PubMed Central

    Zheng, Fengwei; Lu, Guoliang; Li, Ling

    2017-01-01

    ABSTRACT The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a “closed” global conformation related to the NS3-NS4A cis-cleavage event. Although this conformation is incompatible with protease trans-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo. Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis-cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through

  10. Agent-based modeling: case study in cleavage furrow models

    PubMed Central

    Mogilner, Alex; Manhart, Angelika

    2016-01-01

    The number of studies in cell biology in which quantitative models accompany experiments has been growing steadily. Roughly, mathematical and computational techniques of these models can be classified as “differential equation based” (DE) or “agent based” (AB). Recently AB models have started to outnumber DE models, but understanding of AB philosophy and methodology is much less widespread than familiarity with DE techniques. Here we use the history of modeling a fundamental biological problem—positioning of the cleavage furrow in dividing cells—to explain how and why DE and AB models are used. We discuss differences, advantages, and shortcomings of these two approaches. PMID:27811328

  11. Minor dehydrogenated and cleavaged dammarane-type saponins from the steamed roots of Panax notoginseng.

    PubMed

    Gu, Cheng-Zhen; Lv, Jun-Jiang; Zhang, Xiao-Xia; Yan, Hui; Zhu, Hong-Tao; Luo, Huai-Rong; Wang, Dong; Yang, Chong-Ren; Xu, Min; Zhang, Ying-Jun

    2015-06-01

    Nine new minor dehydrogenated and cleavaged dammarane-type triterpenoid saponins, namely notoginsenosides ST6-ST14 (1-9) were isolated from the steamed roots of Panax notoginseng, together with 14 known ones. Among them, 5-7 and 21-22 were protopanaxadiol type and the left 18 compounds, including 1-4, 8-20, and 23 were protopanaxatriol type saponins. Their structures were identified by extensive analysis of MS, 1D and 2D NMR spectra, and acidic hydrolysis. Resulted from the side chain cleavage, the new saponins 1 and 2 featured in a ketone group at C-25, and 3-5 had an aldehyde unit at C-23. The known saponins 12, 16 and 18 displayed the enhancing potential of neurite outgrowth of NGF-mediated PC12 cells at a concentration of 10 μM, while 20 exhibited acetyl cholinesterase inhibitory activity, with IC50 value of 13.97 μM. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Proteomic Analysis of Tendon Extracellular Matrix Reveals Disease Stage-specific Fragmentation and Differential Cleavage of COMP (Cartilage Oligomeric Matrix Protein)*

    PubMed Central

    Dakin, Stephanie Georgina; Smith, Roger Kenneth Whealands; Heinegård, Dick; Önnerfjord, Patrik; Khabut, Areej; Dudhia, Jayesh

    2014-01-01

    During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease. PMID:24398684

  13. Transition State Charge Stabilization and Acid-Base Catalysis of mRNA Cleavage by the Endoribonuclease RelE

    PubMed Central

    Dunican, Brian F.; Hiller, David A.; Strobel, Scott A.

    2015-01-01

    The bacterial toxin RelE is a ribosome-dependent endoribonuclease. It is part of a type II toxin-antitoxin system that contributes to antibiotic resistance and biofilm formation. During amino acid starvation RelE cleaves mRNA in the ribosomal A-site, globally inhibiting protein translation. RelE is structurally similar to microbial RNases that employ general acid-base catalysis to facilitate RNA cleavage. The RelE active-site is atypical for acid-base catalysis, in that it is enriched for positively charged residues and lacks the prototypical histidine-glutamate catalytic pair, making the mechanism of mRNA cleavage unclear. In this study we use a single-turnover kinetic analysis to measure the effect of pH and phosphorothioate substitution on the rate constant for cleavage of mRNA by wild-type RelE and seven active-site mutants. Mutation and thio-effects indicate a major role for stabilization of increased negative change in the transition state by arginine 61. The wild-type RelE cleavage rate constant is pH-independent, but the reaction catalyzed by many of the mutants is strongly pH dependent, suggestive of general acid-base catalysis. pH-rate curves indicate that wild-type RelE operates with the pKa of at least one catalytic residue significantly downshifted by the local environment. Mutation of any single active-site residue is sufficient to disrupt this microenvironment and revert the shifted pKa back above neutrality. pH-rate curves are consistent with K54 functioning as a general base and R81 as a general acid. The capacity of RelE to effect a large pKa shift and facilitate a common catalytic mechanism by uncommon means furthers our understanding of other atypical enzymatic active sites. PMID:26535789

  14. Ideal Cardiovascular Health and Arterial Stiffness in Spanish Adults-The EVIDENT Study.

    PubMed

    García-Hermoso, Antonio; Martínez-Vizcaíno, Vicente; Gomez-Marcos, Manuel Ángel; Cavero-Redondo, Iván; Recio-Rodriguez, José Ignacio; García-Ortiz, Luis

    2018-05-01

    Studies concerning ideal cardiovascular (CV) health and its relationship with arterial stiffness are lacking. This study examined the association between arterial stiffness with ideal CV health as defined by the American Heart Association, across age groups and gender. The cross-sectional study included 1365 adults. Ideal CV health was defined as meeting ideal levels of the following components: 4 behaviors (smoking, body mass index, physical activity, and Mediterranean diet adherence) and 3 factors (total cholesterol, blood pressure, and glycated hemoglobin). Patients were grouped into 3 categories according to their number of ideal CV health metrics: ideal (5-7 metrics), intermediate (3-4 metrics), and poor (0-2 metrics). We analyzed the pulse wave velocity (PWV), the central and radial augmentation indexes, and the ambulatory arterial stiffness index (AASI). The ideal CV health profile was inversely associated with lower arterial radial augmentation index and AASI in both genders, particularly in middle-aged (45-65 years) and in elderly subjects (>65 years). Also in elderly subjects, adjusted models showed that adults with at least 3 health metrics at ideal levels had significantly lower PWV than those with 2 or fewer ideal health metrics. An association was found between a favorable level of ideal CV health metrics and lower arterial stiffness across age groups. Copyright © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  15. Effect of the timing of the first cleavage on the developmental potential of nuclear-transferred mouse oocytes receiving embryonic stem cells.

    PubMed

    Kobayashi, T; Kato, Y; Tsunoda, Y

    2004-09-01

    The present study examined whether the timing of the first cleavage has an effect on the in vitro and in vivo developmental potential of nuclear-transferred mouse oocytes receiving embryonic stem cells. First, the timing of the first cleavage and the developmental potential of nuclear-transferred oocytes were examined every hour from 12 to 24 h after the start of culture and compared with in vitro-fertilized oocytes. The developmental potential of in vitro-fertilized oocytes decreased gradually according to the time required for cleavage (84% (32/38) for 15 h to 50% (1/2) for 20 h), but intermediate-cleaved (15-16 h) nuclear-transferred oocytes had a higher potential to develop into blastocysts (55% (17/31) to 67% (45/67) versus 0-43% (6/14)]. Second the nuclear-transferred oocytes were divided into three groups according to the timing of the first cleavage; each group was cultured to blastocysts in vitro, and then transferred to recipients. The potential of intermediate-cleaved oocytes (15-16 h) to develop into blastocysts was significantly higher than fast-cleaved (before 15 h) and slow-cleaved (after 16 h) oocytes (65, 46, and 37%). The proportion of fetuses on Day 10.5 of pregnancy was highest in the intermediate-cleaved group (4 versus 2 and 1%, respectively) and a full-term fetus was obtained from this group. The present study demonstrated that the timing of the first cleavage could be used to determine the potential of nuclear-transferred oocytes with embryonic stem cells to develop to the blastocyst stage in vitro, but not to determine post-implantation viability after transfer to recipients.

  16. Ideal-Magnetohydrodynamic-Stable Tilting in Field-Reversed Configurations

    NASA Astrophysics Data System (ADS)

    Kanno, Ryutaro; Ishida, Akio; Steinhauer, Loren

    1995-02-01

    The tilting mode in field-reversed configurations (FRC) is examined using ideal-magnetohydrodynamic stability theory. Tilting, a global mode, is the greatest threat for disruption of FRC confinement. Previous studies uniformly found tilting to be unstable in ideal theory: the objective here is to ascertain if stable equilibria were overlooked in past work. Solving the variational problem with the Rayleigh-Ritz technique, tilting-stable equilibria are found for sufficiently hollow current profile and sufficient racetrackness of the separatrix shape. Although these equilibria were not examined previously, the present conclusion is quite surprising. Consequently checks of the method are offered. Even so it cannot yet be claimed with complete certainty that stability has been proved: absolute confirmation of ideal-stable tilting awaits the application of more complete methods.

  17. Cleavage of HPV-16 E6/E7 mRNA mediated by modified 10-23 deoxyribozymes.

    PubMed

    Reyes-Gutiérrez, Pablo; Alvarez-Salas, Luis M

    2009-09-01

    Deoxyribozymes (DXZs) are small oligodeoxynucleotides capable of mediating phosphodiester bond cleavage of a target RNA in a sequence-specific manner. These molecules are a new generation of artificial catalytic nucleic acids currently used to silence many disease-related genes. The present study describes a DXZ (Dz1023-434) directed against the polycistronic mRNA from the E6 and E7 genes of human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer. Dz1023-434 showed efficient cleavage against a bona fide antisense window at nt 410-445 within HPV-16 E6/E7 mRNA even in low [Mg(2+)] conditions. Using a genetic analysis as guidance, we introduced diverse chemical modifications within Dz1023-434 catalytic core to produce a stable locked nucleic acid (LNA)-modified DXZ (Dz434-LNA) with significant cleavage activity of full E6/E7 transcripts. Cell culture testing of Dz434-LNA produced a sharp decrement of E6/E7 mRNA levels in HPV-16-positive cells resulting in decreased proliferation and considerable cell death in a specific and dose-dependent manner. No significant effects were observed with inactive or scrambled control DXZs nor from using HPV-negative cells, suggesting catalysis-dependent effect and high specificity. The biological effects of Dz434-LNA suggest a potential use for the treatment of cervical cancer.

  18. Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

    PubMed Central

    Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

    2016-01-01

    CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

  19. Rock strength measurements on Archaean basement granitoids recovered from scientific drilling in the active Koyna seismogenic zone, western India

    NASA Astrophysics Data System (ADS)

    Goswami, Deepjyoti; Akkiraju, Vyasulu V.; Misra, Surajit; Roy, Sukanta; Singh, Santosh K.; Sinha, Amalendu; Gupta, Harsh; Bansal, B. K.; Nayak, Shailesh

    2017-08-01

    Reservoir triggered earthquakes have been occurring in the Koyna area, western India for the past five decades. Triaxial tests carried out on 181 core samples of Archaean granitoids underlying the Deccan Traps provide valuable constraints on rock strength properties in the Koyna seismogenic zone for the first time. The data include measurements on granite gneiss, granite, migmatitic gneiss and mylonitised granite gneiss obtained from boreholes KBH-3, KBH-4A, KBH-5 and KBH-7 located in the western and eastern margins of the seismic zone. Salient results are as follows. (i) Increase of rock strength with increasing confining pressure allow determination of the linearized failure envelopes from which the cohesive strength and angle of internal friction are calculated. (ii) Variable differential stresses at different depths are the manifestations of deformation partitioning in close association of fault zone(s) or localized fracture zones. (iii) Fractures controlled by naturally developed weak planes such as cleavage and fabric directly affect the rock strength properties, but the majority of failure planes developed during triaxial tests is not consistent with the orientations of pre-existing weak planes. The failure planes may, therefore, represent other planes of weakness induced by ongoing seismic activity. (iv) Stress-strain curves confirm that axial deformation is controlled by the varying intensity of pre-existing shear in the granitoids, viz., mylonite, granite gneiss and migmatitic gneiss. (v) Frequent occurrences of low magnitude earthquakes may be attributed to low and variable rock strength of the granitoids, which, in turn, is modified by successive seismic events.

  20. Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Jinsil; Ha, Hye-Jeong; Kim, Sujin

    Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that anmore » rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.« less

  1. Primitive ideals of C q [ SL(3)

    NASA Astrophysics Data System (ADS)

    Hodges, Timothy J.; Levasseur, Thierry

    1993-10-01

    The primitive ideals of the Hopf algebra C q [ SL(3)] are classified. In particular it is shown that the orbits in Prim C q [ SL(3)] under the action of the representation group H ≅ C *× C * are parameterized naturally by W×W, where W is the associated Weyl group. It is shown that there is a natural one-to-one correspondence between primitive ideals of C q [ SL(3)] and symplectic leaves of the associated Poisson algebraic group SL(3, C).

  2. The strength of a loosely defined movement: eugenics and medicine in imperial Russia.

    PubMed

    Krementsov, Nikolai

    2015-01-01

    This essay examines the 'infiltration' of eugenics into Russian medical discourse during the formation of the eugenics movement in western Europe and North America in 1900-17. It describes the efforts of two Russian physicians, the bacteriologist and hygienist Nikolai Gamaleia (1859-1949) and the psychiatrist Tikhon Iudin (1879-1949), to introduce eugenics to the Russian medical community, analysing in detail what attracted these representatives of two different medical specialties to eugenic ideas, ideals, and policies advocated by their western colleagues. On the basis of a close examination of the similarities and differences in Gamaleia's and Iudin's attitudes to eugenics, the essay argues that lack of cohesiveness gave the early eugenics movement a unique strength. The loose mix of widely varying ideas, ideals, methods, policies, activities and proposals covered by the umbrella of eugenics offered to a variety of educated professionals in Russia and elsewhere the possibility of choosing, adopting and adapting particular elements to their own national, professional, institutional and disciplinary contexts, interests and agendas.

  3. Temperature and the Ideal Gas

    ERIC Educational Resources Information Center

    Daisley, R. E.

    1973-01-01

    Presents some organized ideas in thermodynamics which are suitable for use with high school (GCE A level or ONC) students. Emphases are placed upon macroscopic observations and intimate connection of the modern definition of temperature with the concept of ideal gas. (CC)

  4. Wide-range ideal 2D Rashba electron gas with large spin splitting in Bi2Se3/MoTe2 heterostructure

    NASA Astrophysics Data System (ADS)

    Wang, Te-Hsien; Jeng, Horng-Tay

    2017-02-01

    An application-expected ideal two-dimensional Rashba electron gas, i.e., nearly all the conduction electrons occupy the Rashba bands, is crucial for semiconductor spintronic applications. We demonstrate that such an ideal two-dimensional Rashba electron gas with a large Rashba splitting can be realized in a topological insulator Bi2Se3 ultrathin film grown on a transition metal dichalcogenides MoTe2 substrate through first-principle calculations. Our results show the Rashba bands exclusively over a very large energy interval of about 0.6 eV around the Fermi level within the MoTe2 semiconducting gap. Such a wide-range ideal two-dimensional Rashba electron gas with a large spin splitting, which is desirable for real devices utilizing the Rashba effect, has never been found before. Due to the strong spin-orbit coupling, the strength of the Rashba splitting is comparable with that of the heavy-metal surfaces such as Au and Bi surfaces, giving rise to a spin precession length as small as 10 nm. The maximum in-plane spin polarization of the inner (outer) Rashba band near the Γ point is about 70% (60%). The room-temperature coherence length is at least several times longer than the spin precession length, providing good coherency through the spin processing devices. The wide energy window for ideal Rashba bands, small spin precession length, as well as long spin coherence length in this two-dimensional topological insulator/transition metal dichalcogenides heterostructure pave the way for realizing an ultrathin nano-scale spintronic device such as the Datta-Das spin transistor at room-temperature.

  5. Impaired Cleavage of Preproinsulin Signal Peptide Linked to Autosomal-Dominant Diabetes

    PubMed Central

    Liu, Ming; Lara-Lemus, Roberto; Shan, Shu-ou; Wright, Jordan; Haataja, Leena; Barbetti, Fabrizio; Guo, Huan; Larkin, Dennis; Arvan, Peter

    2012-01-01

    Recently, missense mutations upstream of preproinsulin’s signal peptide (SP) cleavage site were reported to cause mutant INS gene-induced diabetes of youth (MIDY). Our objective was to understand the molecular pathogenesis using metabolic labeling and assays of proinsulin export and insulin and C-peptide production to examine the earliest events of insulin biosynthesis, highlighting molecular mechanisms underlying β-cell failure plus a novel strategy that might ameliorate the MIDY syndrome. We find that whereas preproinsulin-A(SP23)S is efficiently cleaved, producing authentic proinsulin and insulin, preproinsulin-A(SP24)D is inefficiently cleaved at an improper site, producing two subpopulations of molecules. Both show impaired oxidative folding and are retained in the endoplasmic reticulum (ER). Preproinsulin-A(SP24)D also blocks ER exit of coexpressed wild-type proinsulin, accounting for its dominant-negative behavior. Upon increased expression of ER–oxidoreductin-1, preproinsulin-A(SP24)D remains blocked but oxidative folding of wild-type proinsulin improves, accelerating its ER export and increasing wild-type insulin production. We conclude that the efficiency of SP cleavage is linked to the oxidation of (pre)proinsulin. In turn, impaired (pre)proinsulin oxidation affects ER export of the mutant as well as that of coexpressed wild-type proinsulin. Improving oxidative folding of wild-type proinsulin may provide a feasible way to rescue insulin production in patients with MIDY. PMID:22357960

  6. Multilayer regulatory mechanisms control cleavage factor I proteins in filamentous fungi

    PubMed Central

    Rodríguez-Romero, J.; Franceschetti, M.; Bueno, E.; Sesma, A.

    2015-01-01

    Cleavage factor I (CFI) proteins are core components of the polyadenylation machinery that can regulate several steps of mRNA life cycle, including alternative polyadenylation, splicing, export and decay. Here, we describe the regulatory mechanisms that control two fungal CFI protein classes in Magnaporthe oryzae: Rbp35/CfI25 complex and Hrp1. Using mutational, genetic and biochemical studies we demonstrate that cellular concentration of CFI mRNAs is a limited indicator of their protein abundance. Our results suggest that several post-transcriptional mechanisms regulate Rbp35/CfI25 complex and Hrp1 in the rice blast fungus, some of which are also conserved in other ascomycetes. With respect to Rbp35, these include C-terminal processing, RGG-dependent localization and cleavage, C-terminal autoregulatory domain and regulation by an upstream open reading frame of Rbp35-dependent TOR signalling pathway. Our proteomic analyses suggest that Rbp35 regulates the levels of proteins involved in melanin and phenylpropanoids synthesis, among others. The drastic reduction of fungal CFI proteins in carbon-starved cells suggests that the pre-mRNA processing pathway is altered. Our findings uncover broad and multilayer regulatory mechanisms controlling fungal polyadenylation factors, which have profound implications in pre-mRNA maturation. This area of research offers new avenues for fungicide design by targeting fungal-specific proteins that globally affect thousands of mRNAs. PMID:25514925

  7. Structural and Functional Characterization of Cleavage and Inactivation of Human Serine Protease Inhibitors by the Bacterial SPATE Protease EspPα from Enterohemorrhagic E. coli

    PubMed Central

    Weiss, André; Joerss, Hanna; Brockmeyer, Jens

    2014-01-01

    EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition. PMID:25347319

  8. Ideal photon number amplifier and duplicator

    NASA Technical Reports Server (NTRS)

    Dariano, G. M.

    1992-01-01

    The photon number-amplification and number-duplication mechanism are analyzed in the ideal case. The search for unitary evolutions leads to consider also a number-deamplification mechanism, the symmetry between amplification and deamplification being broken by the integer-value nature of the number operator. Both transformations, amplification and duplication, need an auxiliary field which, in the case of amplification, turns out to be amplified in the inverse way. Input-output energy conservation is accounted for using a classical pump or through frequency-conversion of the fields. Ignoring one of the fields is equivalent to considering the amplifier as an open system involving entropy production. The Hamiltonians of the ideal devices are given and compared with those of realistic systems.

  9. Effect of vitamin A deprivation on the cholesterol side-chain cleavage enzyme activity of testes and ovaries of rats (Short Communication)

    PubMed Central

    Jayaram, M.; Murthy, S. K.; Ganguly, J.

    1973-01-01

    The cholesterol side-chain cleavage enzyme activity is decreased considerably at the mild stage of vitamin A deficiency in rat testes and ovaries and the decrease in activity becomes more pronounced with progress of deficiency. Supplementation of the deficient rats with retinyl acetate, but not retinoic acid, restores the enzyme activity to normal values. The cholesterol side-chain cleavage enzyme of adrenals is not affected by any of the above treatments. PMID:4772624

  10. Innovation in neurosurgery: less than IDEAL? A systematic review.

    PubMed

    Muskens, I S; Diederen, S J H; Senders, J T; Zamanipoor Najafabadi, A H; van Furth, W R; May, A M; Smith, T R; Bredenoord, A L; Broekman, M L D

    2017-10-01

    Surgical innovation is different from the introduction of novel pharmaceuticals. To help address this, in 2009 the IDEAL Collaboration (Idea, Development, Exploration, Assessment, Long-term follow-up) introduced the five-stage framework for surgical innovation. To evaluate the framework feasibility for novel neurosurgical procedure introduction, two innovative surgical procedures were examined: the endoscopic endonasal approach for skull base meningiomas (EEMS) and the WovenEndobridge (WEB device) for endovascular treatment of intracranial aneurysms. The published literature on EEMS and WEB devices was systematically reviewed. Identified studies were classified according to the IDEAL framework stage. Next, studies were evaluated for possible categorization according to the IDEAL framework. Five hundred seventy-six papers describing EEMS were identified of which 26 papers were included. No prospective studies were identified, and no studies reported on ethical approval or patient informed consent for the innovative procedure. Therefore, no clinical studies could be categorized according to the IDEAL Framework. For WEB devices, 6229 articles were screened of which 21 were included. In contrast to EEMS, two studies were categorized as 2a and two as 2b. The results of this systematic review demonstrate that both EEMS and WEB devices were not introduced according to the (later developed in the case of EEMS) IDEAL framework. Elements of the framework such as informed consent, ethical approval, and rigorous outcomes reporting are important and could serve to improve the quality of neurosurgical research. Alternative study designs and the use of big data could be useful modifications of the IDEAL framework for innovation in neurosurgery.

  11. Cleavage of Poly(A)-Binding Protein by Enterovirus Proteases Concurrent with Inhibition of Translation In Vitro

    PubMed Central

    Joachims, Michelle; Van Breugel, Pieter C.; Lloyd, Richard E.

    1999-01-01

    Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3′ end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5′-end–3′-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in

  12. Efficient trans-cleavage by the Schistosoma mansoni SMα1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus

    PubMed Central

    Vazquez-Tello, Alejandro; Castán, Pablo; Moreno, Renata; Smith, James M.; Berenguer, José; Cedergren, Robert

    2002-01-01

    The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMα DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme–product dissociation step. The optimal temperature for trans-cleavage was 70°C. This result was confirmed when both the SMα1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMα1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMα1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo. PMID:11917021

  13. Effect of fusion protein cleavage site sequence on generation of a genotype VII Newcastle disease virus vaccine.

    PubMed

    Manoharan, Vinoth K; Varghese, Berin P; Paldurai, Anandan; Samal, Siba K

    2018-01-01

    Newcastle disease (ND) causes severe economic loss to poultry industry worldwide. Frequent outbreaks of ND in commercial chickens vaccinated with live vaccines suggest a need to develop improved vaccines that are genetically matched against circulating Newcastle disease virus (NDV) strains. In this study, the fusion protein cleavage site (FPCS) sequence of NDV strain Banjarmasin/010 (Banj), a genotype VII NDV, was individually modified using primer mutagenesis to those of avian paramyxovirus (APMV) serotypes 2, 7 and 8 and compared with the recombinant Banjarmasin (rBanj) with avirulent NDV LaSota cleavage site (rBanj-LaSota). These FPCS mutations changed the in vitro cell-to-cell fusion activity and made rBanj FPCS mutant viruses highly attenuated in chickens. When chickens immunized with the rBanj FPCS mutant viruses and challenged with the virulent Banj, there was reduced challenge virus shedding observed compared to chickens immunized with the heterologous vaccine strain LaSota. Among the genotype VII NDV Banj vaccine candidates, rBanj-LaSota and rBanj containing FPCS of APMV-8 induced highest neutralizing antibody titers and protected chickens with reduced challenge virus shedding. These results show the effect of the F protein cleavage site sequence in generating genotype VII matched NDV vaccines.

  14. Birth of normal infants after transfer of embryos that were twice vitrified/warmed at cleavage stages: report of two cases.

    PubMed

    Valle, Marcello; Guimarães, Fernando; Cavagnoli, Melissa; Sampaio, Marcos; Geber, Selmo

    2012-12-01

    The role of cryopreservation in assisted reproductive technology programs has increased within the last years allowing the transfer of a limited number of embryos and the storage of the remaining for future use. The reduction in the number of transferred embryos decreases the frequency of multiple pregnancy rates and of ovarian hyperstimulation syndrome while the cumulative pregnancy rate can be maximized. Moreover, as not all embryos will survive the warming process more cleavage stage embryos are warmed to improve selection for transfer. Therefore, surplus good quality cleavage stage embryos and/or blastocysts must be re-vitrified for further transfer to achieve pregnancy. To our knowledge, there have been no reports demonstrating that human embryos can be successfully vitrified/warmed twice at the cleavage stage. Thus we report two successful pregnancies and deliveries of healthy babies after transfer of embryos that were twice vitrified/warmed at 2-4 cells stage. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Elastin: a representative ideal protein elastomer.

    PubMed Central

    Urry, D W; Hugel, T; Seitz, M; Gaub, H E; Sheiba, L; Dea, J; Xu, J; Parker, T

    2002-01-01

    During the last half century, identification of an ideal (predominantly entropic) protein elastomer was generally thought to require that the ideal protein elastomer be a random chain network. Here, we report two new sets of data and review previous data. The first set of new data utilizes atomic force microscopy to report single-chain force-extension curves for (GVGVP)(251) and (GVGIP)(260), and provides evidence for single-chain ideal elasticity. The second class of new data provides a direct contrast between low-frequency sound absorption (0.1-10 kHz) exhibited by random-chain network elastomers and by elastin protein-based polymers. Earlier composition, dielectric relaxation (1-1000 MHz), thermoelasticity, molecular mechanics and dynamics calculations and thermodynamic and statistical mechanical analyses are presented, that combine with the new data to contrast with random-chain network rubbers and to detail the presence of regular non-random structural elements of the elastin-based systems that lose entropic elastomeric force upon thermal denaturation. The data and analyses affirm an earlier contrary argument that components of elastin, the elastic protein of the mammalian elastic fibre, and purified elastin fibre itself contain dynamic, non-random, regularly repeating structures that exhibit dominantly entropic elasticity by means of a damping of internal chain dynamics on extension. PMID:11911774

  16. Mechanism of C-C and C-H bond cleavage in ethanol oxidation reaction on Cu2O(111): a DFT-D and DFT+U study.

    PubMed

    Xu, Han; Miao, Bei; Zhang, Minhua; Chen, Yifei; Wang, Lichang

    2017-10-04

    The performance of transition metal catalysts for ethanol oxidation reaction (EOR) in direct ethanol fuel cells (DEFCs) may be greatly affected by their oxidation. However, the specific effect and catalytic mechanism for EOR of transition metal oxides are still unclear and deserve in-depth exploitation. Copper as a potential anode catalyst can be easily oxidized in air. Thus, in this study, we investigated C-C and C-H bond cleavage reactions of CH x CO (x = 1, 2, 3) species in EOR on Cu 2 O(111) using PBE+U calculations, as well as the specific effect of +U correction on the process of adsorption and reaction on Cu 2 O(111). It was revealed that the catalytic performance of Cu 2 O(111) for EOR was restrained compared with that of Cu(100). Except for the C-H cleavage of CH 2 CO, all the reaction barriers for C-C and C-H cleavage were higher than those on Cu(100). The most probable pathway for CH 3 CO to CHCO on Cu 2 O(111) was the continuous dehydrogenation reaction. Besides, the barrier for C-C bond cleavage increased due to the loss of H atoms in the intermediate. Moreover, by the comparison of the traditional GGA/PBE method and the PBE+U method, it could be concluded that C-C cleavage barriers would be underestimated without +U correction, while C-H cleavage barriers would be overestimated. +U correction was proved to be necessary, and the reaction barriers and the values of the Hubbard U parameter had a proper linear relationship.

  17. Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

    PubMed Central

    Xu, J; Mendez, E; Caron, P R; Lin, C; Murcko, M A; Collett, M S; Rice, C M

    1997-01-01

    Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication. PMID:9188600

  18. Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

    PubMed

    Burruel, Victoria; Klooster, Katie; Barker, Christopher M; Pera, Renee Reijo; Meyers, Stuart

    2014-10-13

    Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (<1 hr) P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

  19. Effects of ionic strength and ion pairing on (plant-wide) modelling of anaerobic digestion.

    PubMed

    Solon, Kimberly; Flores-Alsina, Xavier; Mbamba, Christian Kazadi; Volcke, Eveline I P; Tait, Stephan; Batstone, Damien; Gernaey, Krist V; Jeppsson, Ulf

    2015-03-01

    Plant-wide models of wastewater treatment (such as the Benchmark Simulation Model No. 2 or BSM2) are gaining popularity for use in holistic virtual studies of treatment plant control and operations. The objective of this study is to show the influence of ionic strength (as activity corrections) and ion pairing on modelling of anaerobic digestion processes in such plant-wide models of wastewater treatment. Using the BSM2 as a case study with a number of model variants and cationic load scenarios, this paper presents the effects of an improved physico-chemical description on model predictions and overall plant performance indicators, namely effluent quality index (EQI) and operational cost index (OCI). The acid-base equilibria implemented in the Anaerobic Digestion Model No. 1 (ADM1) are modified to account for non-ideal aqueous-phase chemistry. The model corrects for ionic strength via the Davies approach to consider chemical activities instead of molar concentrations. A speciation sub-routine based on a multi-dimensional Newton-Raphson (NR) iteration method is developed to address algebraic interdependencies. The model also includes ion pairs that play an important role in wastewater treatment. The paper describes: 1) how the anaerobic digester performance is affected by physico-chemical corrections; 2) the effect on pH and the anaerobic digestion products (CO2, CH4 and H2); and, 3) how these variations are propagated from the sludge treatment to the water line. Results at high ionic strength demonstrate that corrections to account for non-ideal conditions lead to significant differences in predicted process performance (up to 18% for effluent quality and 7% for operational cost) but that for pH prediction, activity corrections are more important than ion pairing effects. Both are likely to be required when precipitation is to be modelled. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Topological photonic crystal with ideal Weyl points

    NASA Astrophysics Data System (ADS)

    Wang, Luyang; Jian, Shao-Kai; Yao, Hong

    Weyl points in three-dimensional photonic crystals behave as monopoles of Berry flux in momentum space. Here, based on symmetry analysis, we show that a minimal number of symmetry-related Weyl points can be realized in time-reversal invariant photonic crystals. We propose to realize these ``ideal'' Weyl points in modified double-gyroid photonic crystals, which is confirmed by our first-principle photonic band-structure calculations. Photonic crystals with ideal Weyl points are qualitatively advantageous in applications such as angular and frequency selectivity, broadband invisibility cloaking, and broadband 3D-imaging.

  1. Strength and failure analysis of composite-to-composite adhesive bonds with different surface treatments

    NASA Astrophysics Data System (ADS)

    Paranjpe, Nikhil; Alamir, Mohammed; Alonayni, Abdullah; Asmatulu, Eylem; Rahman, Muhammad M.; Asmatulu, Ramazan

    2018-03-01

    Adhesives are widely utilized materials in aviation, automotive, energy, defense, and marine industries. Adhesive joints are gradually supplanting mechanical fasteners because they are lightweight structures, thus making the assembly lighter and easier. They also act as a sealant to prevent a structural joint from galvanic corrosion and leakages. Adhesive bonds provide high joint strength because of the fact that the load is distributed uniformly on the joint surface, while in mechanical joints, the load is concentrated at one point, thus leading to stress at that point and in turn causing joint failures. This research concentrated on the analysis of bond strength and failure loads in adhesive joint of composite-to-composite surfaces. Different durations of plasma along with the detergent cleaning were conducted on the composite surfaces prior to the adhesive applications and curing processes. The joint strength of the composites increased about 34% when the surface was plasma treated for 12 minutes. It is concluded that the combination of different surface preparations, rather than only one type of surface treatment, provides an ideal joint quality for the composites.

  2. Investigation of the mechanism of meiotic DNA cleavage by VMA1-derived endonuclease uncovers a meiotic alteration in chromatin structure around the target site.

    PubMed

    Fukuda, Tomoyuki; Ohta, Kunihiro; Ohya, Yoshikazu

    2006-06-01

    VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation.

  3. Investigation of the Mechanism of Meiotic DNA Cleavage by VMA1-Derived Endonuclease Uncovers a Meiotic Alteration in Chromatin Structure around the Target Site

    PubMed Central

    Fukuda, Tomoyuki; Ohta, Kunihiro; Ohya, Yoshikazu

    2006-01-01

    VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation. PMID:16757746

  4. Determination of performance of non-ideal aluminized explosives.

    PubMed

    Keshavarz, Mohammad Hossein; Mofrad, Reza Teimuri; Poor, Karim Esmail; Shokrollahi, Arash; Zali, Abbas; Yousefi, Mohammad Hassan

    2006-09-01

    Non-ideal explosives can have Chapman-Jouguet (C-J) detonation pressure significantly different from those expected from existing thermodynamic computer codes, which usually allows finding the parameters of ideal detonation of individual high explosives with good accuracy. A simple method is introduced by which detonation pressure of non-ideal aluminized explosives with general formula C(a)H(b)N(c)O(d)Al(e) can be predicted only from a, b, c, d and e at any loading density without using any assumed detonation products and experimental data. Calculated detonation pressures show good agreement with experimental values with respect to computed results obtained by complicated computer code. It is shown here how loading density and atomic composition can be integrated into an empirical formula for predicting detonation pressure of proposed aluminized explosives.

  5. Bcl2-independent chromatin cleavage is a very early event during induction of apoptosis in mouse thymocytes after treatment with either dexamethasone or ionizing radiation.

    PubMed

    Hahn, Peter J; Lai, Zhi-Wei; Nevaldine, Barbara; Schiff, Ninel; Fiore, Nancy C; Silverstone, Allen E

    2003-11-01

    We have quantified the emergence of early chromatin breaks during the signal transduction phase of apoptosis in mouse thymocytes after treatment with either ionizing radiation or dexamethasone. Dexamethasone at 1 microM can induce significant levels of DNA breaks (equivalent to the amount induced directly by 7.5 Gy ionizing radiation) within 0.5 h of treatment. The execution phase of apoptosis was not observed until 4-6 h after the same treatment. The presence of the Bcl2 transgene under the control of the p56lck promoter almost completely inhibited apoptosis up to 24 h after treatment, but it had virtually no effect on the early chromatin cleavage occurring in the first 6 h. Ionizing radiation induced chromatin cleavage both directly by damaging DNA and indirectly with kinetics similar to the induction of chromatin cleavage by dexamethasone. The presence of the Bcl2 transgene had no effect on the direct or indirect radiation-induced cleavage in the first 6 h, but after the first 6 h, the Bcl2 gene inhibited further radiation-induced chromatin cleavage. These results suggest that endonucleases are activated within minutes of treatment with either dexamethasone or ionizing radiation as part of the very early signal transduction phase of apoptosis, and prior to the irreversible commitment to cell death.

  6. Ultrafast spectroscopy on DNA-cleavage by endonuclease in molecular crowding.

    PubMed

    Singh, Priya; Choudhury, Susobhan; Dutta, Shreyasi; Adhikari, Aniruddha; Bhattacharya, Siddhartha; Pal, Debasish; Pal, Samir Kumar

    2017-10-01

    The jam-packed intracellular environments differ the activity of a biological macromolecule from that in laboratory environments (in vitro) through a number of mechanisms called molecular crowding related to structure, function and dynamics of the macromolecule. Here, we have explored the structure, function and dynamics of a model enzyme protein DNase I in molecular crowing of polyethylene glycol (PEG; MW 3350). We have used steady state and picosecond resolved dynamics of a well-known intercalator ethidium bromide (EB) in a 20-mer double-stranded DNA (dsDNA) to monitor the DNA-cleavage by the enzyme in absence and presence PEG. We have also labelled the enzyme by a well-known fluorescent probe 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) to study the molecular mechanism of the protein-DNA association through exited state relaxation of the probe in absence (dictated by polarity) and presence of EB in the DNA (dictated by Förster resonance energy transfer (FRET)). The overall and local structures of the protein in presence of PEG have been followed by circular dichroism and time resolved polarization gated spectroscopy respectively. The enhanced dynamical flexibility of protein in presence of PEG as revealed from excited state lifetime and polarization gated anisotropy of ANS has been correlated with the stronger DNA-binding for the higher nuclease activity. We have also used conventional experimental strategy of agarose gel electrophoresis to monitor DNA-cleavage and found consistent results of enhanced nuclease activities both on synthetic 20-mer oligonucleotide and long genomic DNA from calf thymus. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Prevalence of ideal cardiovascular health in European adolescents: The HELENA study.

    PubMed

    Henriksson, Pontus; Henriksson, Hanna; Gracia-Marco, Luis; Labayen, Idoia; Ortega, Francisco B; Huybrechts, Inge; España-Romero, Vanesa; Manios, Yannis; Widhalm, Kurt; Dallongeville, Jean; González-Gross, Marcela; Marcos, Ascensión; Moreno, Luis A; Castillo, Manuel J; Ruiz, Jonatan R

    2017-08-01

    The ideal cardiovascular health (iCVH) construct consists of 4 health behaviours and 3 health factors and is strongly related to later cardiovascular disease. However, the prevalence of iCVH in European adolescents is currently unknown. The Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) study is a cross-sectional, multicentre study conducted in 9 European countries during 2006-2007 and included 3528 adolescents (1683 boys and 1845 girls) between 12.5 and 17.5years of age. Status (ideal vs. non-ideal) for the health behaviours (smoking status, body mass index, physical activity and diet) and health factors (total cholesterol, blood pressure and fasting glucose) were determined. Overall, the prevalence of ideal health behaviours was low; non-smoking (60.9% ideal), body mass index (76.8%), physical activity (62.1%), and diet (1.7%). The prevalence of ideal health factors was; total cholesterol (65.8%), blood pressure (62.0%) and plasma glucose (88.8%). The low prevalence of iCVH behaviours, especially diet and physical activity, identified in European adolescents is likely to influence later cardiovascular health which strongly motivates efforts to increase ideal health behaviours in this population. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Ideal affect in daily life: implications for affective experience, health, and social behavior.

    PubMed

    Tsai, Jeanne L

    2017-10-01

    Over the last decade, researchers have increasingly demonstrated that ideal affect-the affective states that people value and ideally want to feel-shapes different aspects of daily life. Here I briefly review Affect Valuation Theory (AVT), which integrates ideal affect into existing models of affect and emotion by identifying the causes and consequences of variation in ideal affect. I then describe recent research that applies AVT to the valuation of negative states as well as more complex states, examines how ideal affect shapes momentary affective experience, suggests that ideal affect has both direct and indirect effects on health, and illustrates that people's ideal affect shapes how they judge and respond to others. Finally, I discuss the implications of cultural and individual differences in ideal affect for clinical, educational, work, and leisure settings. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Mechanistic Insights into Archaeal and Human Argonaute Substrate Binding and Cleavage Properties

    PubMed Central

    Willkomm, Sarah; Zander, Adrian; Grohmann, Dina; Restle, Tobias

    2016-01-01

    Argonaute (Ago) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these Ago proteins, among them human Argonaute2 (hAgo2) and Ago from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an Ago-associated complementary guide strand. Here we present an in-depth kinetic side-by-side analysis of hAgo2 and MjAgo guide and target substrate binding as well as target strand cleavage, which enabled us to disclose similarities and differences in the mechanistic pathways as a function of the chemical nature of the substrate. Testing all possible guide-target combinations (i.e. RNA/RNA, RNA/DNA, DNA/RNA and DNA/DNA) with both Ago variants we demonstrate that the molecular mechanism of substrate association is highly conserved among archaeal-eukaryotic Argonautes. Furthermore, we show that hAgo2 binds RNA and DNA guide strands in the same fashion. On the other hand, despite striking homology between the two Ago variants, MjAgo cannot orientate guide RNA substrates in a way that allows interaction with the target DNA in a cleavage-compatible orientation. PMID:27741323

  10. Raman characterization of Avocado Sunblotch viroid and its response to external perturbations and self-cleavage

    PubMed Central

    2014-01-01

    Background Viroids are the smallest pathogens of plants. To date the structural and conformational details of the cleavage of Avocado sunblotch viroid (ASBVd) and the catalytic role of Mg2+ ions in efficient self-cleavage are of crucial interest. Results We report the first Raman characterization of the structure and activity of ASBVd, for plus and minus viroid strands. Both strands exhibit a typical A-type RNA conformation with an ordered double-helical content and a C3′-endo/anti sugar pucker configuration, although small but specific differences are found in the sugar puckering and base-stacking regions. The ASBVd(-) is shown to self-cleave 3.5 times more actively than ASBVd(+). Deuteration and temperature increase perturb differently the double-helical content and the phosphodiester conformation, as revealed by corresponding characteristic Raman spectral changes. Our data suggest that the structure rigidity and stability are higher and the D2O accessibility to H-bonding network is lower for ASBVd(+) than for ASBVd(-). Remarkably, the Mg2+-activated self-cleavage of the viroid does not induce any significant alterations of the secondary viroid structure, as evidenced from the absence of intensity changes of Raman marker bands that, however exhibit small but noticeable frequency downshifts suggesting several minor changes in phosphodioxy, internal loops and hairpins of the cleaved viroids. Conclusions Our results demonstrate the sensitivity of Raman spectroscopy in monitoring structural and conformational changes of the viroid and constitute the basis for further studies of its interactions with therapeutic agents and cell membranes. PMID:24655924

  11. Atorvastatin prevents Aβ oligomer-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting Tau cleavage

    PubMed Central

    Sui, Hai-juan; Zhang, Ling-ling; Liu, Zhou; Jin, Ying

    2015-01-01

    Aim: The proteolytic cleavage of Tau is involved in Aβ-induced neuronal dysfunction and cell death. In this study, we investigated whether atorvastatin could prevent Tau cleavage and hence prevent Aβ1–42 oligomer (AβO)-induced neurotoxicity in cultured cortical neurons. Methods: Cultured rat hippocampal neurons were incubated in the presence of AβOs (1.25 μmol/L) with or without atorvastatin pretreatment. ATP content and LDH in the culture medium were measured to assess the neuronal viability. Caspase-3/7 and calpain protease activities were detected. The levels of phospho-Akt, phospho-Erk1/2, phospho-GSK3β, p35 and Tau proteins were measured using Western blotting. Results: Treatment of the neurons with AβO significantly decreased the neuronal viability, induced rapid activation of calpain and caspase-3/7 proteases, accompanied by Tau degradation and relatively stable fragments generated in the neurons. AβO also suppressed Akt and Erk1/2 kinase activity, while increased GSK3β and Cdk5 activity in the neurons. Pretreatment with atorvastatin (0.5, 1, 2.5 μmol/L) dose-dependently inhibited AβO-induced activation of calpain and caspase-3/7 proteases, and effectively diminished the generation of Tau fragments, attenuated synaptic damage and increased neuronal survival. Atorvastatin pretreatment also prevented AβO-induced decreases in Akt and Erk1/2 kinase activity and the increases in GSK3β and Cdk5 kinase activity. Conclusion: Atorvastatin prevents AβO-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting calpain- and caspase-mediated Tau cleavage. PMID:25891085

  12. C-terminal N-alkylated peptide amides resulting from the linker decomposition of the Rink amide resin: a new cleavage mixture prevents their formation.

    PubMed

    Stathopoulos, Panagiotis; Papas, Serafim; Tsikaris, Vassilios

    2006-03-01

    Decomposition of the resin linkers during TFA cleavage of the peptides in the Fmoc strategy leads to alkylation of sensitive amino acids. The C-terminal amide alkylation, reported for the first time, is shown to be a major problem in peptide amides synthesized on the Rink amide resin. This side reaction occurs as a result of the Rink amide linker decomposition under TFA treatment of the peptide resin. The use of 1,3-dimethoxybenzene in a cleavage cocktail prevents almost quantitatively formation of C-terminal N-alkylated peptide amides. Oxidized by-product in the tested Cys- and Met-containing peptides were not observed, even if thiols were not used in the cleavage mixture. Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.

  13. Commercialization of NASA's High Strength Cast Aluminum Alloy for High Temperature Applications

    NASA Technical Reports Server (NTRS)

    Lee, Jonathan A.

    2003-01-01

    In this paper, the commercialization of a new high strength cast aluminum alloy, invented by NASA-Marshall Space Flight Center, for high temperature applications will be presented. Originally developed to meet U.S. automotive legislation requiring low- exhaust emission, the novel NASA aluminum alloy offers dramatic improvement in tensile and fatigue strengths at elevated temperatures (450 F-750 F), which can lead to reducing part weight and cost as well as improving performance for automotive engine applications. It is an ideal low cost material for cast components such as pistons, cylinder heads, cylinder liners, connecting rods, turbo chargers, impellers, actuators, brake calipers and rotors. NASA alloy also offers greater wear resistance, dimensional stability, and lower thermal expansion compared to conventional aluminum alloys, and the new alloy can be produced economically from sand, permanent mold and investment casting. Since 2001, this technology was licensed to several companies for automotive and marine internal combustion engines applications.

  14. Cardiorespiratory fitness and ideal cardiovascular health in European adolescents.

    PubMed

    Ruiz, Jonatan R; Huybrechts, Inge; Cuenca-García, Magdalena; Artero, Enrique G; Labayen, Idoia; Meirhaeghe, Aline; Vicente-Rodriguez, German; Polito, Angela; Manios, Yannis; González-Gross, Marcela; Marcos, Ascensión; Widhalm, Kurt; Molnar, Denes; Kafatos, Anthony; Sjöström, Michael; Moreno, Luis A; Castillo, Manuel J; Ortega, Francisco B

    2015-05-15

    We studied in European adolescents (i) the association between cardiorespiratory fitness and ideal cardiovascular health as defined by the American Heart Association and (ii) whether there is a cardiorespiratory fitness threshold associated with a more favourable cardiovascular health profile. Participants included 510 (n=259 girls) adolescents from 9 European countries. The 20 m shuttle run test was used to estimate cardiorespiratory fitness. Ideal cardiovascular health was defined as meeting ideal levels of the following components: four behaviours (smoking, body mass index, physical activity and diet) and three factors (total cholesterol, blood pressure and glucose). Higher levels of cardiorespiratory fitness were associated with a higher number of ideal cardiovascular health components in both boys and girls (both p for trend ≤0.001). Levels of cardiorespiratory fitness were significantly higher in adolescents meeting at least four ideal components (13% higher in boys, p<0.001; 6% higher in girls, p=0.008). Receiver operating characteristic curve analyses showed a significant discriminating accuracy of cardiorespiratory fitness in identifying the presence of at least four ideal cardiovascular health components (43.8 mL/kg/min in boys and 34.6 mL/kg/min in girls, both p<0.001). The results suggest a hypothetical cardiorespiratory fitness level associated with a healthier cardiovascular profile in adolescents. The fitness standards could be used in schools as part of surveillance and/or screening systems to identify youth with poor health behaviours who might benefit from intervention programmes. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  15. Non-ideal Solution Thermodynamics of Cytoplasm

    PubMed Central

    Ross-Rodriguez, Lisa U.; McGann, Locksley E.

    2012-01-01

    Quantitative description of the non-ideal solution thermodynamics of the cytoplasm of a living mammalian cell is critically necessary in mathematical modeling of cryobiology and desiccation and other fields where the passive osmotic response of a cell plays a role. In the solution thermodynamics osmotic virial equation, the quadratic correction to the linear ideal, dilute solution theory is described by the second osmotic virial coefficient. Herein we report, for the first time, intracellular solution second osmotic virial coefficients for four cell types [TF-1 hematopoietic stem cells, human umbilical vein endothelial cells (HUVEC), porcine hepatocytes, and porcine chondrocytes] and further report second osmotic virial coefficients indistinguishable from zero (for the concentration range studied) for human hepatocytes and mouse oocytes. PMID:23840923

  16. Ideal Standards, Acceptance, and Relationship Satisfaction: Latitudes of Differential Effects

    PubMed Central

    Buyukcan-Tetik, Asuman; Campbell, Lorne; Finkenauer, Catrin; Karremans, Johan C.; Kappen, Gesa

    2017-01-01

    We examined whether the relations of consistency between ideal standards and perceptions of a current romantic partner with partner acceptance and relationship satisfaction level off, or decelerate, above a threshold. We tested our hypothesis using a 3-year longitudinal data set collected from heterosexual newlywed couples. We used two indicators of consistency: pattern correspondence (within-person correlation between ideal standards and perceived partner ratings) and mean-level match (difference between ideal standards score and perceived partner score). Our results revealed that pattern correspondence had no relation with partner acceptance, but a positive linear/exponential association with relationship satisfaction. Mean-level match had a significant positive association with actor’s acceptance and relationship satisfaction up to the point where perceived partner score equaled ideal standards score. Partner effects did not show a consistent pattern. The results suggest that the consistency between ideal standards and perceived partner attributes has a non-linear association with acceptance and relationship satisfaction, although the results were more conclusive for mean-level match. PMID:29033876

  17. A statistical model of brittle fracture by transgranular cleavage

    NASA Astrophysics Data System (ADS)

    Lin, Tsann; Evans, A. G.; Ritchie, R. O.

    A MODEL for brittle fracture by transgranular cleavage cracking is presented based on the application of weakest link statistics to the critical microstructural fracture mechanisms. The model permits prediction of the macroscopic fracture toughness, KI c, in single phase microstructures containing a known distribution of particles, and defines the critical distance from the crack tip at which the initial cracking event is most probable. The model is developed for unstable fracture ahead of a sharp crack considering both linear elastic and nonlinear elastic ("elastic/plastic") crack tip stress fields. Predictions are evaluated by comparison with experimental results on the low temperature flow and fracture behavior of a low carbon mild steel with a simple ferrite/grain boundary carbide microstructure.

  18. Disclosure of key stereoelectronic factors for efficient H2 binding and cleavage in the active site of [NiFe]-hydrogenases.

    PubMed

    Bruschi, Maurizio; Tiberti, Matteo; Guerra, Alessandro; De Gioia, Luca

    2014-02-05

    A comparative analysis of a series of DFT models of [NiFe]-hydrogenases, ranging from minimal NiFe clusters to very large systems including both the first and second coordination sphere of the bimetallic cofactor, was carried out with the aim of unraveling which stereoelectronic properties of the active site of [NiFe]-hydrogenases are crucial for efficient H2 binding and cleavage. H2 binding to the Ni-SIa redox state is energetically favored (by 4.0 kcal mol(-1)) only when H2 binds to Ni, the NiFe metal cluster is in a low spin state, and the Ni cysteine ligands have a peculiar seesaw coordination geometry, which in the enzyme is stabilized by the protein environment. The influence of the Ni coordination geometry on the H2 binding affinity was then quantitatively evaluated and rationalized analyzing frontier molecular orbitals and populations. Several plausible reaction pathways leading to H2 cleavage were also studied. It turned out that a two-step pathway, where H2 cleavage takes place on the Ni-SIa redox state of the enzyme, is characterized by very low reaction barriers and favorable reaction energies. More importantly, the seesaw coordination geometry of Ni was found to be a key feature for facile H2 cleavage. The discovery of the crucial influence of the Ni coordination geometry on H2 binding and activation in the active site of [NiFe]-hydrogenases could be exploited in the design of novel biomimetic synthetic catalysts.

  19. Developmental Idealism, Body Weight and Shape, and Marriage Entry in Transitional China.

    PubMed

    Xu, Hongwei

    2016-04-01

    New trends toward later and less marriage are emerging in post-reform China. Previous research has examined the changing individual-level socioeconomic and demographic characteristics shaping marriage entry in Chinese adults. Employing a cultural model known as developmental idealism (DI), this study argues that a new worldview specifying an ideal body type has become popular in the West and that this new worldview has been exported to China. This new part of the DI package is likely stratified by gender, has a stronger impact on women than on men, and has likely penetrated urban areas more than rural areas. Drawing on the 1991-2009 longitudinal data from the China Health and Nutrition Survey, this study employs discrete-time logit models to estimate the relationships between various body types and transition to first marriage in Chinese young adults 18-30 years old. Body weight status and body shape are measured by body mass index (BMI) and waist-to-hip ratio (WHR), respectively, and further divided into categories of underweight, normal, and obese. Regression results indicate that larger values of BMI and WHR were associated with delayed entry into first marriage in urban women, whereas being overweight or obese was associated with accelerated transition to first marriage in rural men. Not only were these associations statistically significant, but their strengths were substantively remarkable. Findings from this study suggest that both body weight and body shape have important implications for marital success, independent of individual-level socioeconomic and demographic characteristics, and contribute to evolving gender and rural-urban disparities, as China is undergoing a rapid nutrition transition.

  20. Developmental Idealism, Body Weight and Shape, and Marriage Entry in Transitional China

    PubMed Central

    Xu, Hongwei

    2016-01-01

    New trends toward later and less marriage are emerging in post-reform China. Previous research has examined the changing individual-level socioeconomic and demographic characteristics shaping marriage entry in Chinese adults. Employing a cultural model known as developmental idealism (DI), this study argues that a new worldview specifying an ideal body type has become popular in the West and that this new worldview has been exported to China. This new part of the DI package is likely stratified by gender, has a stronger impact on women than on men, and has likely penetrated urban areas more than rural areas. Drawing on the 1991-2009 longitudinal data from the China Health and Nutrition Survey, this study employs discrete-time logit models to estimate the relationships between various body types and transition to first marriage in Chinese young adults 18-30 years old. Body weight status and body shape are measured by body mass index (BMI) and waist-to-hip ratio (WHR), respectively, and further divided into categories of underweight, normal, and obese. Regression results indicate that larger values of BMI and WHR were associated with delayed entry into first marriage in urban women, whereas being overweight or obese was associated with accelerated transition to first marriage in rural men. Not only were these associations statistically significant, but their strengths were substantively remarkable. Findings from this study suggest that both body weight and body shape have important implications for marital success, independent of individual-level socioeconomic and demographic characteristics, and contribute to evolving gender and rural-urban disparities, as China is undergoing a rapid nutrition transition. PMID:27909585

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