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  • Articles: DFG German National Licenses  (2,016)
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  • 101
    Electronic Resource
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 33-50 
    ISSN: 0029-5981
    Keywords: finite element method ; shape optimization ; sensitivity analysis ; forming process ; optimal design ; forging ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: We suggest a shape optimization method for a non-linear and non-steady-state metal forming problem. It consists in optimizing the initial shape of the part as well as the shape of the preform tool during a two-step forging operation, for which the shape of the second operation is known. Shapes are described using spline functions and optimal parameter values of the splines are searched in order to produce, at the end of the forging sequence, a part with a prescribed geometric accuracy, optimal metallurgical properties and for a minimal production cost. The finite element method, including numerous remeshing operations, is used for the simulation of the process. We suggest using a least-squares-type algorithm for the unconstrained optimization method (based on external penalty) for which we describe the calculation of the derivatives of the objective function. We show that it can reduce to calculations which are equivalent to the derivative calculations of steady-state processes and to evolution equations. Therefore, the computational cost of such an optimization is quite reasonable, even for complex forging processes. Lastly, in order to reduce the errors due to the numerous remeshings during the simulation, we introduce error estimation and adaptive remeshing methods with respect to the calculation of derivatives.
    Additional Material: 6 Ill.
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  • 102
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 165-184 
    ISSN: 0029-5981
    Keywords: curved unilateral contact ; augmented Lagrangian ; frictional contact element ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: Based on a mixed formulation approach, a frictional contact element is proposed for the numerical solution of contact problems including strongly curved rigid obstacles. The implementation of the frictional contact element is analogous to that of a finite element. This feature facilitates its implementation in implicit finite element programmes, since the structure of the code need not be modified.For efficient modelling of the forming tool geometries by Computer Aided Geometric Design techniques and in order to achieve a high performance of the contact search, the numerical schemes of the frictional contact element operate directly on parametric polynomial surface patches. Thus, no discretization of curved contact surfaces is performed.Numerical simulations of deep drawing processes demonstrate the performance of the method in the case of large sliding increments upon curved tools and in the case of elasto-plasticity.
    Additional Material: 8 Ill.
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  • 103
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1097-1114 
    ISSN: 0029-5981
    Keywords: finite elements ; pneumatics ; air springs ; containers ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: Many engineering structures completely surround and enclose gas filled volumes. The enclosed gas adds additional stiffness to the surrounding structure. This paper shows how to account for this effect by means of an augmented virtual work principle. The additional term augmenting the virtual work equilibrium statement for the structure is the virtual boundary work done by the pressure of the enclosed gas. The augmented equations are discretized using standard finite element methods, and the additional terms are discussed. The resulting ‘pneumatic’ finite element is shown to be analogous to regular structural finite elements. To assess the accuracy and efficiency and also to illustrate the applicability of the present formulation, a series of four examples was selected. In two of the examples, the behaviour of the end cap of a partially filled plastic food product container is studied. The numerical results using the pneumatic element compare well with an alternative Rayleigh-Ritz solution of the end cap behaviour. The other two examples represent the behaviour of a double bellows air spring shock absorber under static isothermal and dynamic adiabatic conditions. For the static isothermal case, an experimental study was performed with results in good agreement with the pneumatic element solutions. For the dynamic adiabatic loading case, the dynamic stiffness of the air spring was predicted using the pneumatic element. The numerical results agree with experimental data published in an air spring application guide. The examples illustrate that the pneumatic element formulation can be applied to the large deflection analysis of structures that enclose gas filled volumes.
    Additional Material: 9 Ill.
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  • 104
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1115-1136 
    ISSN: 0029-5981
    Keywords: particle tracking ; Lagrangian-Eulerian finite element methods ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper presents a multi-dimensional particle tracking technique for applying the Lagrangian-Eulerian finite element method to solve transport equations in transient-state simulations. In the Lagrangian- Eulerian approach, the advection term is handled in the Lagrangian step so that the associated numerical errors can be considerably reduced. It is important to have an adequate particle tracking technique for computing advection accurately in the Lagrangian step. The particle tracking technique presented here is designed to trace fictitious particles in the real-world flow field where the flow velocity is either measured or computed at a limited number of locations. The technique, named ‘in-element’ particle tracking, traces fictitious particles on an element-by-element basis. Given a velocity field, a fictitious particle is traced one element by one element until either a boundary is encountered or the available time is completely consumed. For the tracking within an element, the element is divided into a desired number of subelements with the interpolated velocity computed at all nodes of the subelements. A fictitious particle, thus, is traced one subelement by one subelement within the element. The desired number of subelements can be determined based on the complexity of the flow field being considered. The more complicated the flow field is, the more subelements are needed to achieve accurate particle tracking results. A single-velocity approach can be used to efficiently perform particle tracking in a smooth flow field, while an average-velocity approach can be employed to increase the tracking accuracy for more complex flow fields.
    Additional Material: 15 Ill.
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  • 105
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1313-1340 
    ISSN: 0029-5981
    Keywords: iterative methods for linear systems ; preconditioning ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The preconditioned conjugate gradient algorithm is a well-known and powerful method used to solve large sparse symmetric positive definite linear systems. Such systems are generated by the finite element discretization in structural analysis but users of finite elements in this context generally still rely on direct methods. It is our purpose in the present work to highlight the improvement brought forward by some new preconditioning techniques and show that the preconditioned conjugate gradient method performs better than efficient direct methods.
    Additional Material: 8 Ill.
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  • 106
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1341-1356 
    ISSN: 0029-5981
    Keywords: constrained optimization ; feasible descent ; mathematical programming ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A new Feasible Descent Cone (FDC) method for constrained optimization, previously restricted to linear objectives, is here generalized to include non-linear objective functions as well. In the basic and exact algorithm a sequence of descent steps is taken through the interior of the feasible region along the central lines of mathematically defined descent cones, constructed at successive boundary points. Here the basic algorithm is modified to allow for a minimum to occur within the interior, along a central descent ray in the case of non-linear objectives. A special interior procedure, with desirable mathematical properties, is adopted should the latter occur. To ensure economic implementation, the new generalized and exact algorithm, referred to as SSOPT2, is successively applied to a sequence of approximate quadratic subproblems. The overall generalized procedure that includes the successive application of SSOPT2 to the approximate subproblems, is referred to as the successive approximation version 2 algorithm (SAM2). The practical performance of SAM2 is assessed through its application to a number of small but otherwise representative test problems.
    Additional Material: 2 Ill.
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  • 107
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1357-1382 
    ISSN: 0029-5981
    Keywords: numerical reduced minimization ; non-uniform isoparametric mapping ; unmatched coefficient ; spurious constraint ; optimal minimization ; locking ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: In this paper, the effects of various numerical integrations on the behaviour of C0-continuous beam elements under non-uniform isoparametric mapping are investigated by using numerical reduced minimization theory. The theory shows that stress recovery can be achieved by sampling stresses at the optimal integration points once a reduced integration is employed. It rationalizes the continued acceptance of the conventional reduced integration of constrained strain energy as one of remedies for locking due to spurious constraint.
    Additional Material: 12 Ill.
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  • 108
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1383-1403 
    ISSN: 0029-5981
    Keywords: crashworthiness ; homogenization ; topology design ; automotive structure optimization ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The topological optimization of components to maximize crash energy absorption for a given volume is considered. The crash analysis is performed using a DYNA3D finite element analysis. The original solid elements are replaced by ones with holes, the hole size being characterized by a so-called density (measure of the reduced volume). A homogenization method is used to find elastic moduli as a function of this density. Simpler approximations were developed to find plastic moduli and yield stress as functions of density.Optimality criteria were derived from an optimization statement using densities as the design variables. A resizing algorithm was constructed so that the optimality criteria are approximately satisfied. A novel feature is the introduction of an objective function based on strain energies weighted at specified times. Each different choice of weighting factors leads to a different structure, allowing a range of design possibilities to be explored.The method was applied to an automotive body rear rail. The original design and a new design of equal volume with holes were compared for energy absorption.
    Additional Material: 25 Ill.
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  • 109
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1419-1431 
    ISSN: 0029-5981
    Keywords: integral equations ; scattering ; boundary elements ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper describes a boundary integral equation (boundary element) method for the solution of a variety of transient acoustic problems. The spatial and temporal discretization employs quadratic isoparametric elements with high-order Gauss quadrature, and the ensuing equations are implicit. The implicit formulation both eliminates the instabilities reported in explicit treatments, and permits a freedom of choice of timestep which can reduce costs dramatically. The accuracy of the approach is demonstrated by comparison with the analytical solution for a sphere. Results for more demanding sphere-cone-sphere geometries extending to seven wavelengths long are presented, and compared to those obtained from a related frequency domain approach.
    Additional Material: 7 Ill.
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  • 110
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1405-1417 
    ISSN: 0029-5981
    Keywords: RTM ; control volume ; free surface ; porous medium ; sparse matrix ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: An efficient numerical scheme is presented for simulating isothermal flow in resin transfer molding. The problem involves transient, free surface flow of an incompressible fluid into a non-deforming porous medium. A new variant of the Control Volume Finite Element (CVFE) algorithm is explained in detail. It is shown how the pressure solutions at each time step can be obtained by adding a single row and column to the Cholesky factorization of the stiffness matrix derived from a finite element formulation for the pressure field. This approach reduces the computation of a new pressure solution at each time step to essentially just two sparse matrix back-substitutions. The resulting performance improvement facilitates interactive simulation and the solution of inverse problems which require many simulations of the filling problem. The computational complexity of the calculation is bounded by O(n2⋅5), where n is the number of nodes in the finite element mesh. A 100-fold speedup over a conventional CVFE implementation was obtained for a 2213-node problem.
    Additional Material: 4 Ill.
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  • 111
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996) 
    ISSN: 0029-5981
    Keywords: Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
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  • 112
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1436-1436 
    ISSN: 0029-5981
    Keywords: Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
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  • 113
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1433-1435 
    ISSN: 0029-5981
    Keywords: Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
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  • 114
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1437-1454 
    ISSN: 0029-5981
    Keywords: large elastic deformations ; enhanced strain method ; finite elements ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The numerical simulation of processes undergoing finite deformations requires robust elements. For a broad range of applications these elements should have a good performance in bending dominated situations as well as in the case of incompressibility. The element should be insensitive against mesh distortions which frequently occurs during finite deformations. Furthermore, due to efficiency reasons a good coarse mesh accuracy in required in non-linear analysis. The QS6 element, developed in this paper, tries to fulfil the above-mentioned requirements. The performance is depicted by means of numerical examples.
    Additional Material: 10 Ill.
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  • 115
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1455-1473 
    ISSN: 0029-5981
    Keywords: transient loads ; advective-diffusive equations ; Petrov-Galerkin ; Galerkin Leask-Square ; boundary layers ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A Petrov-Galerkin formulation based on two different perturbations to the weighting functions is presented. These perturbations stabilize the oscillations that are normally exhibited by the numerical solution of the transient advective-diffusive equation in the vicinity of sharp gradients produced by transient loads and boundary layers. The formulation may be written as a generalization of the Galerkin Least-Square method.
    Additional Material: 8 Ill.
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  • 116
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 1475-1489 
    ISSN: 0029-5981
    Keywords: meshing ; three-dimensional ; surfaces ; quadrilateral ; finite element ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper discusses the extension of the paving algorithm for all quadrilateral mesh generation to arbitrary three-dimensional trimmed surfaces. Methods of calculating angles, projecting elements, and detecting collisions between paving boundaries, for general surfaces are presented. Extensions of the smoothing algorithms for three dimensions are set forth. Advances in the use of scalar sizing functions are presented. These functions can be used to better approximate internal mesh density from boundary densities and surface characteristics.
    Additional Material: 16 Ill.
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  • 117
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 2155-2168 
    ISSN: 0029-5981
    Keywords: buckling ; composite laminated structures ; high-order element ; locking ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A new high-order element for prebuckling and buckling of any laminated plane frame is developed. The kinematic approach involves a variety of high-order models with different powers of the thickness co-ordinate, and the derived stiffness matrix is based on a special power series, without a numerical process. It yields an exact formulation for high-order (first) models and improved formulations for higher-order (second and third) ones. A parametric study of the ‘locking’ phenomenon and the shear deformation effects was carried out for isotropic and laminated structures. It was found that the first-order model with an appropriate shear correction factor yielded results close to its higher-order counterparts.
    Additional Material: 10 Ill.
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  • 118
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 2265-2282 
    ISSN: 0029-5981
    Keywords: energy-conservation ; dynamics ; beams ; co-rotation ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The paper describes an energy-conserving procedure for the implicit non-linear dynamic analysis of planar beam structures. The method is based on a form of co-rotational technique which is ‘external to the element’. A range of numerical results are presented which clearly demonstrate the improved numerical performance in comparison with more conventional techniques.
    Additional Material: 8 Ill.
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  • 119
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 2555-2573 
    ISSN: 0029-5981
    Keywords: 3-D boundary element method ; elastoplastic analysis ; hypersingular formulation ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A 3-D hypersingular Boundary Integral Equation (BIE) of elastoplasticity is derived. Using this formulation the displacement rate gradients and the complete stress tensor on the boundary can be evaluated directly as opposed to the classical approach, where the shape functions derivatives are to be calculated. The regularization of strongly singular and hypersingular boundary integrals, as well as strongly singular domain integrals for a source point positioned on the boundary is carried out in a general manner. Arbitrary types of elements and arbitrary positions of the source point with respect to continuity requirements can be used. Numerical 3-D elastoplastic examples (notch and crack problems) illustrate the advantages of the proposed method.
    Additional Material: 16 Ill.
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  • 120
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 2337-2361 
    ISSN: 0029-5981
    Keywords: elastoplastic ; high-accuracy ; locking-free ; low-order ; nonlinear ; three-field functional ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper presents a family of low-order high-accuracy three-dimensional brick elements. The elements are formulated via a three-field variational principle. The assumed (independent) strain field is constructed from two disjointed distributions. The first contains the lower-order distribution and its dimension is the minimum required to satisfy stability requirements. An energy constraint, which is enforced weakly at the element level, is used to relate the second distribution to the first. The stress field is chosen to a priori satisfy a similar energy constraint. As a result, internal constraints (e.g. incompressibility) are automatically satisfied by these fields, and locking behaviour is avoided. A J2-plasticity model illustrates the proposed elements' performance in nonlinear solids. The excellent performance of the proposed elements is demonstrated with numerous challenging examples, including many that are usually modelled by shell elements.
    Additional Material: 20 Ill.
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  • 121
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996) 
    ISSN: 0029-5981
    Keywords: Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
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  • 122
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 2507-2508 
    ISSN: 0029-5981
    Keywords: Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
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  • 123
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 2531-2554 
    ISSN: 0029-5981
    Keywords: boundary element method ; elastohydrodynamic lubrication ; layered contact ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper presents a numerical routine to compute the contact characteristics of elastomer layered cylinders lubricated by isoviscous liquids. The indentation of the elastic layer is calculated from boundary integral equations which are solved by linear and quadratic boundary element methods for a finite plane model and a circular representation of the junction. The hydrodynamic equation is also transformed into a boundary integral equation and solved by Simpson's rule. Some factors which possibly affect numerical accuracy are examined. Examples for finite plane and circular layer are analysed with reference to parameters for printing press roller contact, in which results are obtained for the indentation, film thickness and liquid pressure, as well as internal stresses through the simultaneous solution of the elasticity and hydrodynamic equations. The results show that high precision is easily achieved and the method is efficient for such layered problems.
    Additional Material: 10 Ill.
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  • 124
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 2789-2802 
    ISSN: 0029-5981
    Keywords: finite elements ; three-dimensional elasticity ; equilibrium formulations ; hybrid finite elements ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: In this paper an approach to the formulation of equilibrium elements for the analysis of three-dimensional elasticity problems is presented.This formulation is an extension of the approach previously proposed for the analysis of two-dimensional elasticity problems. The general aspects of the formulation remain unchanged when applied to the new problem, but new points are considered, namely the way to perform volume integrations for general elements and the techniques used to obtain the self-equilibrated three-dimensional stress approximation functions.The numerical behaviour of such elements is presented and discussed.
    Additional Material: 5 Ill.
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  • 125
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 2837-2862 
    ISSN: 0029-5981
    Keywords: special boundary integral formulation ; design sensitivity analysis (DSA) ; direct differentiation approach (DDA) ; three-dimensional conduction heat transfer ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A special boundary integral formulation had been proposed to analyse many engineering problems of conduction heat transfer in complex three-dimensional geometries (closely spaced surface and circular hole in infinite domain or simple modification of it) by Rezayat and Burton. One example of such geometries is the mold sets in the injection molding process. In this paper, an efficient and accurate approach for the design sensitivity analysis (DSA) is presented for these kinds of problems in the similar complex geometries using the direct differentiation approach (DDA) based on the above special boundary integral formulation. The present approach utilizes the implicit differentiation of the boundary integral equations with respect to the design variables (radii and locations of circular holes) to yield the sensitivity equations. A sample problem (heat transfer of injection molding cooling system) is solved to demonstrate the accuracy of the present sensitivity analysis formulation. Although the techniques introduced here are applied to a particular problem in heat transfer of injection molding cooling system, their potential application is quite broad.
    Additional Material: 14 Ill.
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  • 126
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 2921-2944 
    ISSN: 0029-5981
    Keywords: wavelet orthogonal bases ; wavelet-Galerkin method ; Burgers' equation ; numerical method ; approximation ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper describes exact evaluations of various finite integrals whose integrands involve products of Daubechies' compactly supported wavelets and their derivatives and/or integrals. These finite integrals play an essential role in the wavelet-Galerkin approximation of differential or integral equations on a bounded interval.
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  • 127
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 2973-2985 
    ISSN: 0029-5981
    Keywords: co-rotational frame ; convected bases ; integration of the spin ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A new algorithm is proposed for integrating the spin in the frame of large deformation analysis. The method is based on the integration of a matrix relation, obtained from an adapted decomposition of the deformation gradient, and directly written in convected co-ordinates. The input of this algorithm is either the Gram matrix at any time (if a kinematical method is used, for instance) or more generally, the incremental deformation gradient, and the output is the required rotation matrix on convected bases. The result takes a very simple form in the important case of classical shells.
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  • 128
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    International Journal for Numerical Methods in Engineering 39 (1996) 
    ISSN: 0029-5981
    Keywords: Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 129
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal for Numerical Methods in Engineering 39 (1996), S. 3195-3215 
    ISSN: 0029-5981
    Keywords: modified effective capacitance ; solidification ; tetrahedral elements ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: In this paper a new formulation for modelling solidification is discussed. The formulation has similar features to both the apparent and effective heat capacitance methods used for solidification problems where conduction predominates over other heat transfer mechanisms. The main feature of the new method is that a modified form of effective heat capacitance is calculated from the solution of non-linear equations that describe the energy loss for linear tetrahedral finite elements. This approach ensures that the predicted temperature field corresponds exactly with the energy loss and so providing an extremely stable formulation. The method is tested against a range of problems including some with non-linear liquid fractions. The predictions are compared against known analytical solutions and the method is shown to provide reasonable accuracy even for relatively large time-steps. A comparison is made between the method and the well-known temporal and spatial approximations of apparent heat capacitance, and effective capacitance. Accuracy is maintained over a greater variation in time-step and mesh density with comparable computational requirements. In addition, the method lends itself to the use of relatively simple bisection techniques for the solution of the non-linear finite element equations. Also demonstrated is the method's innate ability to predict energy loss to a high degree of accuracy for large time steps.
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  • 130
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3049-3064 
    ISSN: 0029-5981
    Keywords: mold cooling ; injection molding ; finite element method ; three-dimensional ; Jacobi conjugate gradient ; matrix-free algorithm ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A methodology is presented to simulate the three-dimensional heat transfer within a mold during the injection molding process. The mold cooling analysis assists cooling channel design and paves the way for part shrinkage and warpage analysis. The transient temperature distributions in the mold and the polymer part are simultaneously computed by Galerkin Finite Element Method (GFEM) using a matrix-free Jacobi Conjugate Gradient (JCG) scheme. The numerical method presented here is efficient and has shown to require a fraction of the memory and computing time required by conventional methods. The matrix-free algorithm is initially validated using an injection mold designed to produce a plaque with a molded-in hole. Subsequently, the method is further applied to a representative automotive plastic component.
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  • 131
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 2907-2920 
    ISSN: 0029-5981
    Keywords: sensitivity analysis ; error estimates ; mesh refinement ; hypersingular integrals ; boundary element method ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The subject of this paper is the sensitivity analysis of approximate boundary element solutions with respect to the positions of the collocation points. The direct differentiation approach is considered here and the analysis is performed analytically. Since only the collocation points are perturbed, the shape of the body and the corresponding discretization remain unaltered. This aspect makes the present work quite different in spirit with respect to earlier analyses on shape sensitivities. Sensitivities of approximate BEM solutions with respect to the positions of collocation points are shown to be related to the residual of hypersingular integral equations. Numerical results confirm that the present approach can be seen as the analytical counterpart of an adaptive scheme for mesh refinement presented by the same author in some recent papers. Some other advantages of the present approach over the former one are also outlined.
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  • 132
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3217-3217 
    ISSN: 0029-5981
    Keywords: Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
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  • 133
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3327-3349 
    ISSN: 0029-5981
    Keywords: mesh generation ; hexahedral ; whisker weaving ; duality ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper introduces a new algorithm called whisker weaving for constructing unstructured, all-hexahedral finite element meshes. Whisker weaving is based on the Spatial Twist Continuum (STC), a global interpretation of the geometric dual of an all-hexahedral mesh. Whisker weaving begins with a closed, all-quadrilateral surface mesh bounding a solid geometry, then constructs hexahedral element connectivity advancing into the solid. The result of the whisker weaving algorithm is a complete representation of hex mesh connectivity only: Actual mesh node locations are determined afterwards.The basic step of whisker weaving is to form a hexahedral element by crossing or intersecting dual entities. This operation, combined with seaming or joining operations in dual space, is sufficient to mesh simple block problems. When meshing more complex geometries, certain other dual entities appear such as blind chords, merged sheets, and self-intersecting chords. Occasionally specific types of invalid connectivity arise. These are detected by a general method based on repeated STC edges. This leads into a strategy for resolving some cases of invalidities immediately.The whisker weaving implementation has so far been successful at generating meshes for simple block-type geometries and for some non-block geometries. Mesh sizes are currently limited to a few hundred elements. While the size and complexity of meshes generated by whisker weaving are currently limited, the algorithm shows promise for extension to much more general problems.
    Additional Material: 25 Ill.
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  • 134
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3367-3390 
    ISSN: 0029-5981
    Keywords: finite plasticity ; single crystals ; multisurface plasticity ; active set search ; exponential map ; stress update algorithm ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper presents a new stress update algorithm for large-strain rate-independent single-crystal plasticity. The theoretical frame is the well-established continuum slip theory based on the multiplicative decomposition of the deformation gradient into elastic and plastic parts. A distinct feature of the present formulation is the introduction and computational exploitation of a particularly simple hyperelastic stress response function based on a further multiplicative decomposition of the elastic deformation gradient into spherical and unimodular parts, resulting in a very convenient representation of the Schmid resolved shear stresses on the crystallographic slip systems in terms of a simple inner product of Eulerian vectors. The key contribution of this paper is an algorithmic formulation of the exponential map exp: sl(3) → SL(3) for updating the special linear group SL(3) of unimodular plastic deformation maps. This update preserves exactly the plastic incompressibility condition of the anisotropic plasticity model under consideration. The resulting fully implicit stress update algorithm treats the possibly redundant constraints of single-crystal plasticity by means of an active set search. It exploits intrinsically the simple representation of the Schmid stresses by formulating the return algorithm and the associated consistent elastoplastic moduli in terms of Eulerian vectors updates. The performance of the proposed algorithm is demonstrated by means of a representative numerical example.
    Additional Material: 10 Ill.
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  • 135
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    International Journal for Numerical Methods in Engineering 39 (1996) 
    ISSN: 0029-5981
    Keywords: Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
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  • 136
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3663-3677 
    ISSN: 0029-5981
    Keywords: matrix condition ; heat equation ; discontinuous coefficients ; error stability ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The traditional tridiagonal matrix approximating the one-dimensional heat equation is ill-conditioned when heat conductivity changes radically. An algebraic reformulation of the tridiagonal produces a well-conditioned matrix. Additional variables are rates q=-kux at interfaces between radical changes in k. A reduced matrix amounts to a coarse approximation.
    Additional Material: 4 Ill.
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  • 137
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3679-3703 
    ISSN: 0029-5981
    Keywords: 3-D fracture ; interface ; boundary element ; singular integration ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: Mixed-mode fractures of arbitrary orientation with respect to a planar bimaterial interface have been effectively modelled using a surface integral approach. By requiring only that the surface of the fracture be discretized, the surface integral method circumvents the practical difficulties associated with having to mesh the interacting dual singularities in stress along the three-dimensional (3-D) crack front and at the interface. The key elements of this numerical capability are discussed in detail. These include: the derivation of the fundamental solutions for a generalized fracture event near a planar bimaterial interface, formulation of the governing integral equation including its decomposition into singular and non-singular terms, development of analytical and numerical techniques for performing the singular integrations, and efficient numerical integration of the non-singular terms using non-dimensionalized surface approximations of the dipole solutions. The problem of a pressurized planar crack near a bimaterial interface was used to assess convergence. The effect of material contrast and crack shape on tendencies for crack growth were also examined.
    Additional Material: 17 Ill.
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  • 138
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3453-3474 
    ISSN: 0029-5981
    Keywords: plate ; finite element ; locking ; non-linear ; buckling ; vibrations ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A new eight-node C0 membrane-plate quadrilateral finite element is presented to analyse static and dynamic moderately large deflections of moderately thick laminates. The finite element is based on the Reissner- Mindlin plate theory and on previous works for the shear-bending (plate) part, which has been described in a previous paper, Reference 10. In the paper, the novelty is in the way of improving isoparametric membrane response without adding zero-energy modes, and the moderately large deflection static and dynamic analysis of laminates based on von Karman's assumptions. Finally, some numerical simulations are presented in statics and in dynamics for laminates, including buckling analysis and membrane-plate coupling effects. Among a large variety of finite elements which have been proposed during the two last decades, interest in the present element lies in its simplicity implementation, its efficiency without any correction factors and spurious energy modes, and (also) the fact it is fully standard from an engineering view point.
    Additional Material: 10 Ill.
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  • 139
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3475-3495 
    ISSN: 0029-5981
    Keywords: structural dynamics ; time finite elements ; Hermitian shape functions ; unconditionally stable algorithms ; higher-order accurate algorithms ; time-step integration ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: In this paper, single step time finite elements using the cubic Hermitian shape functions to interpolate the solution over a time interval are considered. The second-order differential equations are manipulated directly. Both the effects of modal damping and external excitation are considered. The accuracy of the solutions at the end of the time interval and the interpolated solutions within the time interval is investigated. The weighted residual approach is adopted to derive the time-integration algorithms. Instead of specifying the weighting functions, the weighting parameters are used to control the characteristics of the time finite elements. The weighting parameters are chosen to eliminate the higher-order truncation error terms or to enforce the asymptotic annihilation condition. A one-parameter family of third-order accurate asymptotically annihilating algorithms and another one-parameter family of fourth-order accurate non-dissipative algorithms are presented. The ranges of the weighting parameters for unconditionally stable algorithms are given. It is found that one of the members in each family corresponds to the Padé approximants of the exponential function in solving the first-order differential equations. Some of the existing unconditionally stable higher-order accurate algorithms are re-derived by the present unified approach.
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  • 140
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    International Journal for Numerical Methods in Engineering 39 (1996) 
    ISSN: 0029-5981
    Keywords: Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
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  • 141
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3553-3569 
    ISSN: 0029-5981
    Keywords: finite/infinite element method ; infinite element ; semi-infinite problem ; soil-structure interaction ; wave propagation ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: Two drawbacks exist with the infinite elements used for simulating the unbounded domains of semi-infinite problems. The first is the lack of an adequate measure for calculating the decay parameter. The second is the frequency-dependent characteristic of the finite/infinite element mesh used for deriving the impedance matrices. Based on the properties of wave propagation, a scheme is proposed in this paper for evaluating the decay parameter. In addition, it is shown that by the method of dynamic condensation, the far-field impedance matrices for waves of lower frequencies can be obtained repetitively from the one for waves of the highest frequency, using exactly the same finite/infinite element mesh. Such an approach ensures that accuracy of the same degree can be maintained for waves of all frequencies within the range of consideration. Effectiveness of the proposed method is demonstrated in the numerical examples through comparison with previous results.
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  • 142
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3867-3887 
    ISSN: 0029-5981
    Keywords: discrete Kirchhoff theory ; shell element ; semiloof element ; non-linear shell analysis ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: In the formulation of flat shell elements it is difficult to achieve inter-element compatibility between membrane and transverse displacements for non-coplanar elements. Many elements lack proper nodal degrees of freedom to model intersections making the assembly of elements troublesome. A flat triangular shell element is established by a combination of a new plate bending element DKTL and the well-known linear membrane strain element LST, and for this element the above-mentioned deficiencies are avoided. The plate bending element DKTL is based on Discrete Kirchhoff Theory and Loof nodes. The nodal configuration of the element is similar to the SemiLoof element, and the formulation is an improvement of a previous formulation. The element is used for both linear statics, linear buckling and geometrical non-linear analysis, and numerical examples are presented to show the robustness, accuracy and quick convergence of the element.
    Additional Material: 14 Ill.
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  • 143
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3931-3931 
    ISSN: 0029-5981
    Keywords: Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
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  • 144
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3985-3998 
    ISSN: 0029-5981
    Keywords: structural dynamics ; time stepping ; collocation ; Gauss points ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: When a cubic function is interpolated between the prescribed initial displacement and velocity and the exact displacement and velocity at the end of a time step for a single degree of freedom system, the error, or residual, in the governing equation is zero at a number of times. It is shown that for a general undamped system, in the limit as the time step approaches zero these times correspond to Gauss points. This observation is verified by considering a general collocation procedure in which the displacement in any time step is approximated as a cubic function of time, with two coefficients chosen to satisfy the displacement and velocity at the beginning of the time step with the other two coefficients being chosen to satisfy the governing differential equation at any two times. It is shown that optimum accuracy is obtained if these points are the Gauss points. Detailed expressions are then presented for this particular case, and stability of the algorithm is investigated showing that the procedure is conditionally stable. For time steps which are a small proportion of the least period of vibration of the structure, the algorithm is considered to be the most accurate possible procedure based on cubic approximation of the displacement.
    Additional Material: 3 Ill.
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  • 145
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 3999-4020 
    ISSN: 0029-5981
    Keywords: sparse matrices ; frontal methods ; Gaussian elimination ; finite-element equations ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The multiple front algorithm is an extension of the frontal method to allow parallelism to be exploited in the solution process. The finite-element domain is partitioned into a number of subdomains and a frontal decomposition is performed on each subdomain separately. For a given partitioning of the domain, the efficiency of the multiple front algorithm depends on the ordering of the elements within each subdomain. We look at the limitations of existing element reordering algorithms when applied to a subdomain and consider how these limitations may be overcome. Extensive numerical experiments are performed on a range of practical problems and, on the basis of the results, we propose a new element resequencing algorithm for use with a multiple front algorithm.
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  • 146
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    International Journal for Numerical Methods in Engineering 39 (1996), S. 4039-4061 
    ISSN: 0029-5981
    Keywords: finite element method ; optimal mesh ; strategy ; refinement ; error estimation ; Engineering ; Engineering General
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: Five Adaptive Modification Strategies (AMSs) are studied as means to obtain nearly optimal meshes using an adaptive finite element modelling system based on the h-version of the FEM. These strategies include the method traditionally seem in the literature as well as four additional methods. The five strategies are tested over five numerical examples, one of them producing convergence oscillations in one example. The principal characteristic of our system is that it permits the user to control certain parameters of the adaptive process, which contributes to an improvement in the results.
    Additional Material: 11 Ill.
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  • 147
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    Journal of Cellular Biochemistry 60 (1996), S. 4-11 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Genetic analysis of programmed cell death in Caenorhabditis elegans has led to the identification of 13 genes that constitute a developmental pathway of programmed cell death. Two of the three key genes in this pathway, ced-9, a cell death suppressor, and ced-3, a cell death inducer, were found to encode proteins that share structural and functional similarities with the mammalian proto-oncogene product Bcl-2 and interleukin-1β converting enzyme, respectively. These results suggest that the genetic pathway of programmed cell death may be evolutionarily conserved from worms to mammals. © 1996 Wiley-Liss, Inc.
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  • 148
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    Journal of Cellular Biochemistry 60 (1996), S. 12-17 
    ISSN: 0730-2312
    Keywords: bcl-2 gene ; localization ; apoptosis ; antioxidants ; oxidative stress ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The bcl-2 gene has a unique function among mammalian oncogenes as a negative regulator of apoptosis. Its expression pattern in embryonic and adult tissues is consistent with a role in maintaining in vivo survival of specific cell types.The biochemical function of bcl-2 is unknown, but its localization to mitochondrial and microsomal membranes suggests several possibilities, bcl-2 is protective against oxidative stress in mammalian cells and can be replaced by antioxidants in a factor-deprivation model of apoptosis. These results are consistent with a model of apoptotic death involving oxidative stress in a central pathway.The recent discovery of several bcl-2-related genes, some of which also inhibit apoptosis and others that unexpectedly promote apoptosis, has shed new light on several aspects of bcl-2 action. © 1996 Wiley-Liss, Inc.
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  • 149
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    Journal of Cellular Biochemistry 60 (1996), S. 33-38 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: No abstract.
    Additional Material: 3 Ill.
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  • 150
    ISSN: 0730-2312
    Keywords: BCL-2 gene ; Bcl-2 protein ; homologs ; homo- and heterotypic dimers ; cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The BCL-2 gene was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in lymphomas, which result in deregulation of BCL-2 gene expression and cause inappropriately high levels of Bcl-2 protein production. Expression of the BCL-2 gene can also become altered in human cancers through other mechanisms, including loss of the p53 tumor suppressor which normally functions as a repressor of BCL-2 gene expression in some tissues. Bcl-2 is a blocker of programmed cell death and apoptosis that contributes to neoplastic cell expansion by preventing cell turnover caused by physiological cell death mechanisms, as opposed to accelerating rates of cell division. Overproduction of the Bcl-2 protein also prevents cell death induced by nearly all cytotoxic anticancer drugs and radiation, thus contributing to treatment failures in patients with some types of cancer. Several homologs of Bcl-2 have recently been discovered, some of which function as inhibitors of cell death and others as promoters of apoptosis that oppose the actions of the Bcl-2 protein. Many of these Bcl-2 family proteins can interact through formation of homo- and heterotypic dimers. In addition, several nonhomologous proteins have been identified that bind to Bcl-2 and that can modulate apoptosis. These protein-protein interactions may eventual serve as targets for pharmacologically manipulating the physiological cell death pathway for treatment of cancer and several other diseases. © 1996 Wiley-Liss, Inc.
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  • 151
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    Journal of Cellular Biochemistry 60 (1996), S. 61-82 
    ISSN: 0730-2312
    Keywords: protein kinases ; cyclins ; nuclear import ; NLS ; acidic domains ; cell cycle ; phosphatases ; p34cdc2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Karyophilic and acidic clusters were found in most nonmembrane serine/threonine protein kinases whose primary structure was examined. These karyophilic clusters might mediate the anchoring of the kinase molecules to transporter proteins for their regulated nuclear import and might constitute the nuclear localization signals (NLS) of the kinase molecules. In contrast to protein transcription factors that are exclusively nuclear possessing strong karyophilic peptides composed of at least four arginines (R) and lysines (K) within an hexapeptide flanked by proline and glycine helix-breakers, protein kinases often contain one histidine and three K + R residues; this is proposed to specify a weak NLS structure resulting in the nuclear import of a fraction of the total cytoplasmic kinase molecules as well as in their weak retention in the different ionic strength nuclear environment. Putative NLS peptides in protein kinases may also contain hydrophobic or bulky aromatic amino acids proposed to further diminish their capacity to act as strong NLS. Most kinases lacking karyophilic clusters (c-Mos, v-Mos, sea star MAP, and yeast KIN28, SRA1, SRA3, TPK1, TPK2) also lack acidic clusters, which is in contrast to most kinases containing both acidic and karyophilic peptides; this and the presence of R/K clusters in the transporter proteins supports a role of acidic clusters on kinases in nuclear import. Cyclins B lack karyophilic signals and are proposed to be imported into nuclei via their association with Cdc2. © 1996 Wiley-Liss, Inc.
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  • 152
    ISSN: 0730-2312
    Keywords: protein kinase FA/GSK-3α ; PKC inhibition ; calphostin C ; down-regulation ; carcinoma dedifferentiation/progression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The signal transduction mechanism of protein kinase FA/GSK-3α by tyrosine phosphorylation in A431 cells was investigated using calphostin C as an inhibitor for protein kinase C (PKC). Kinase Fa/GSK-3α could be tyrosine-dephosphorylated and inactivated to ∼ 10% of control in a concentration-dependent manner by 0.1-10 μM calphostin C (IC50, ∼ 1 μM), as demonstrated by immunoprecipitation of kinase Fa/GSK-3α from cell extracts, followed by phosphoamino acid analysis and by immunodetection in an antikinase Fa/GSK-3α immunoprecipitate kinase assay. In sharp contrast, down-regulation of PKC by 0.05 μM calphostin C (IC50, ∼ 0.05 μM for inhibiting PKC in cells) or by tumor promoter phorbol ester TPA was found to have stimulatory effect on the cellular activity of kinase Fa/GSK-3α, when processed under identical conditions. Furthermore, TPA-mediated down-regulation of PKC was found to have no effect on calphostin C-mediated tyrosine dephosphorylation/inactivation of kinase Fa/GSK-3α. Taken together, the results provide initial evidence that the PKC inhibitor calphostin C may induce tyrosine dephosphorylation/inactivation of kinase Fa/GSK-3α in a pathway independent of TPA-mediated down-regulation of PKC, representing a new mode of signal transduction for the regulation of this multisubstrate/multifunctional protein kinase by calphostin C in cells. Since kinase Fa/GSK-3α is a possible carcinoma dedifferentiation/progression-promoting factor, the results further suggest calphostin C as a potential anticancer drug involved in blocking carcinoma dedifferentiation/progression, possibly via inactivation of protein kinase FA/GSK-3α in tumor cells. © 1996 Wiley-Liss, Inc.
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  • 153
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    Journal of Cellular Biochemistry 60 (1996), S. 363-378 
    ISSN: 0730-2312
    Keywords: cyclin D1 function ; CDK activity ; pRB phosphorylation ; G1 phase ; cell cycle control ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The sequential transcriptional activation of cyclins, the regulatory subunits of cell cycle specific kinases, regulates progress through the cell cycle. In mitogen-stimulated cells cyclin D1 induction in early G1 is followed by induction of cyclin E, activation of the cyclin-dependent kinase Cdk2, and hyperphosphorylation of the retinoblastoma gene product (pRB) in mid-to-late G1 phase. T-47D breast cancer cells expressing cyclin D1 under the control of a metal-responsive metallothionein promoter were used to determine whether Cdk2 activation and pRB hyperphosphorylation are consequences of cyclin D1 induction. A 4-5-fold increase in cyclin D1 protein abundance was followed by approximately 2-fold increases in cyclin E protein abundance and Cdk2 activity and by hyperphosphorylation of pRB. These responses were apparent ∼ 3 h after the increase in cyclin D1 protein, and ∼ 3 h prior to the entry of cyclin D1-stimulated cells into S phase 12 h after zinc treatment. Cyclin D1 immunoprecipitates contained Cdk4 but no detectable Cdk2 and displayed pRb but not histone H1 kinase activity. Cdk2 activation was therefore likely to be due to increased abundance of cyclin E/Cdk2 complexes rather than formation of active cyclin D1/Cdk2 complexes. The sequence of events following zinc induction of cyclin D1 thus mimicked that following mitogen induction of cyclin D1. These data show that cyclin D1 induction is sufficient for Cdk2 activation and pRB hyperphosphorylation in T-47D human breast cancer cells, providing evidence that cyclin D1 induction is a critical event in G1 phase progression. © 1996 Wiley-Liss, Inc.
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  • 154
    ISSN: 0730-2312
    Keywords: heregulin ; transformation ; erb B-2 ; c-Ha-ras ; mammary cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Heregulin β1 was found to stimulate the anchorage-dependent, serum-free growth of nontransformed human MCF-10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor α, or amphiregulin, heregulin β1 was also able to induce the anchorage-independent growth of MCF-10A cells. In contrast, the anchorage-dependent, serum-free growth of c-Ha-ras or c-erb B-2 transformed MCF-10A cells was unaffected by heregulin β1, whereas heregulin β1 was able to stimulate the anchorage-independent growth of these cells. c-Ha-ras or c-erb B-2 (c-neu) transformed MCF-10A or mouse NOG-8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell-associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF-10A cells and transformants also express c-erb B-3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha-ras or erb B-2 transformed mammary epithelial cells. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.
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  • 155
    ISSN: 0730-2312
    Keywords: ecto-enzyme ; ALP inhibitor ; Ca incorporation ; glycosylphosphatidylinositol-anchored proteins ; PI-PLC ; bone differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p-nitrophenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6-8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP were close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole was uncompetitive (Ki = 45 μM). Phosphatidylinositol-specific phospholipase C (PI-PLC) produced the release into the medium of a p-nitrophenyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The released activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto-pNPPase activity. After 75 min of PI-PLC treatment the ecto-pNPPase activity remained unchanged despite the 30% decrease in Nonidet P-40-extractable ALP activity. High levels of 45Ca incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in accordance with ecto-pNPPase activity variations. These results evidenced the levamisole-sensitive activity of a glycosylphosphatidylinositol-anchored pNPPase consistent with ALP acting as an ecto-enzyme whose functioning under physiological conditions was correlated to 45Ca incorporation and permit the prediction of the physiological importance of the enzyme dynamic equilibrium at the cell surface in cultured fetal calvaria cells. © 1996 Wiley-Liss, Inc.
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  • 156
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    Journal of Cellular Biochemistry 60 (1996), S. 521-528 
    ISSN: 0730-2312
    Keywords: myosin heavy chains ; smooth muscle ; alternative splicing ; contractility ; myosin light chains ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The aim of our study was to determine the relation between alternatively spliced myosin heavy chain (MHC) isoforms and the contractility of smooth muscle. The relative amount of MHC with an alternatively spliced insert in the 5′ (amino terminal) domain was determined on the protein level using a peptide-directed antibody (a25K/50K) raised against the inserted sequence (QGPSFAY). Smooth muscle MHC isoforms of both bladder and myometrium but not nonmuscle MHC reacted with a25/50K. Using a quantitative Western-blot approach the amount of 5′-inserted MHC in rat bladder was detected to be about eightfold higher than in normal rat myometrium. The amount of heavy chain with insert was found to be decreased by about 50% in the myometrium of pregnant rats. Although bladder contained significantly more 5′-inserted MHC than myometrium, apparent maximal shortening velocities (Vmax) were comparable, being 0.138 ± 0.012 and 0.114 ± 0.023 muscle length per second of skinned bladder and normal myometrium fibers, respectively. Phosphorylation of myosin light chain 20 induced by maximal Ca2+/calmodulin activation was the same in bladder and myometrial fibers. These results suggest that the amount of 5′-inserted MHC is not necessarily associated with contractile properties of smooth muscle. © 1996 Wiley-Liss, Inc.
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  • 157
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    Journal of Cellular Biochemistry 61 (1996), S. 9-17 
    ISSN: 0730-2312
    Keywords: antiestrogen ; human breast cancer ; programmed cell death ; tamoxifen ; TGF-β1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-β1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-β was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-β. © 1996 Wiley-Liss, Inc.
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  • 158
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    Journal of Cellular Biochemistry 61 (1996), S. 39-47 
    ISSN: 0730-2312
    Keywords: α2M ; PAF ; RBF ; PKC ; lyso-PAF acetyltransferase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The binding of receptor-recognized forms of α2-macroglobulin (α2M) to macrophage α2M signaling receptors increases inositol-1,4,5-triphosphate synthesis and induces Ca2+ mobilization. In this report, we demonstrate that ligation of the macrophage α2M signaling receptor is also associated with synthesis of platelet activating factor (PAF) by both the de novo and remodeling pathways. Both α2M-methylamine and a cloned and expressed 20-kDa receptor binding fragment (RBF) from rat α2M+, stimulated macrophage synthesis of PAF from [3H]acetate, [3H]methylcholine, and 1-O-[3H]alkyl lyso-PAF by two- to threefold. PAF levels reached a peak in 20 min after the cells were exposed to α2M-methylamine or RBF; they remained elevated for about 1 h after ligand addition to the cells. When [3H]methylcholine was the substrate, pertussis toxin did not block PAF synthesis, but the protein kinase C inhibitor staurosporin reduced synthesis by 65-70%. Cycloheximide completely abolished the increase in synthesis of PAF by macrophages exposed to α2M-methylamine. By contrast, when [3H]acetate was employed as a precursor, staurosporin or cycloheximide did not abolish the increase in PAF synthesis. These studies suggest that protein kinase C is necessary for the induction of the de novo pathway by α2M-methylamine. Both α2M-methylamine and RBF stimulated the activity of lyso-PAF acetyltransferase by about fourfold. Both ligands also stimulated the activity of PAF acetylhydrolase by about six- to sevenfold, indicating that ligation of the α2M signaling receptor also regulates the degradation of PAF. The ability of receptor-recognized forms of α2M to regulate levels of PAF suggests that α2M-proteinase complexes not only regulate macrophage function by activating intracellular signaling but also may indirectly regulate the function of other cells that cannot bind α2M-proteinase complexes. © 1996 Wiley-Liss, Inc.
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  • 159
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    Journal of Cellular Biochemistry 61 (1996), S. 109-117 
    ISSN: 0730-2312
    Keywords: aggregin ; chemical modification ; ADP-induced platelet responses ; NBD-Cl ; cAMP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Additional Material: 5 Ill.
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  • 160
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    Journal of Cellular Biochemistry 61 (1996), S. 72-80 
    ISSN: 0730-2312
    Keywords: hypoxia ; S-adenosylmethionine ; DNA methylation ; hypomethylation ; t-RNA methyltransferase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Young rats were maintained in a 10% oxygen atmosphere for 2, 6, and 10 days and administered normal rat chow and water ad libitum. Thereafter, their hepatic S-adenosyl-L-methionine (AdoMet) and activity and mRNA levels of AdoMet synthetase were assayed. AdoMet levels decreased by 45% after 10 days; hepatic AdoMet synthetase also declined by ∼40%. In rats with low hepatic AdoMet, the mRNA level of AdoMet synthetase also declined by up to 80%. No significant change in AdoMet or AdoMet synthetase was noted in pair-fed normoxic rats. DNA hypomethylation was determined in terms of incorporation of [3H]methyl of AdoMet incorporated at unmethylated sites in DNA in reactions mediated by methylases Hpall and Sssl. As compared to the normal hepatic DNA, [3H]methyl group incorporation in the 10-day hypoxic DNA was almost double in the Hpall-mediated reaction and ∼10-fold in the Sssl-mediated reaction. Hepatic tRNA methyltransferase activity doubled after 10 days of hypoxia. However, hypoxic rats showed no detectable mRNA transcripts for c-myc and c-fos oncogenes on Northern blot analysis. These observations show that because of subnormal activity of AdoMet synthetase, hypoxic liver is depleted of AdoMet, even when the animals are administered a complete diet. However, unlike rats on chronic lipotrope-deficient diets, hypoxic rats on a complete diet show no aberrant expression of oncogenes. © 1996 Wiley-Liss, Inc.
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  • 161
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    Journal of Cellular Biochemistry 61 (1996), S. 31-38 
    ISSN: 0730-2312
    Keywords: cell density ; DNA synthesis ; Mr receptor substrates ; IRS-1 protein ; tyrosine phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In order to examine alterations in the phosphorylation state of proteins involved in insulin action that might accompany the reduced growth state of density-arrested cells, we measured the insulin-stimulated phosphorylation of the receptor and high Mr cellular substrates of the receptor kinase in rat hepatoma cells at different cell densities. As cell density increased from 2 × 105 to 3.2 × 106 per 35-mm well, the rate of DNA synthesis fell to 22% of control, while insulin-stimulated tyrosine phosphorylation of high Mr receptor substrates (“pp185”) was enhanced to 198% of control, without a change in the abundance of insulin receptor substrate (IRS)-1 protein. In anti-IRS-1 immunoprecipitates, tyrosine phosphorylation was increased by only 30%, suggesting that increased tyrosine phosphorylation of additional high Mr proteins (e.g., IRS-2) accounted for much of the observed increase in tyrosine phosphorylation of the receptor substrates. In spite of increased tyrosine phosphorylation of IRS-1 and total pp185-related proteins, however, cells studied at high growth density exhibited a 25% decrease in IRS-1-associated phosphatidylinositol 3′-kinase activity and only a 39% increase in phosphatidylinositol 3′-kinase activity in antiphosphotyrosine immunoprecipitates. To explore the potential role of hepatic protein-tyrosine phosphatases (PTPases) in the hyperphosphorylation of pp185 proteins, we found by immunoblotting that at high cell density the intracellular PTPase PTP18 and the transmembrane PTPase LAR were reduced in abundance by 49% and 55%, respectively, while the abundance of the SH2-domain containing PTPase SH-PTP2 was increased by 48%. These data demonstrate that the attenuation of post-receptor signaling by insulin in hepatoma cells at increasing growth density involves changes in endogenous substrate phosphorylation which may result from alterations in specific PTPases implicated in the regulation of the insulin action pathway. © 1996 Wiley-Liss, Inc.
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  • 162
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    Journal of Cellular Biochemistry 61 (1996), S. 48-60 
    ISSN: 0730-2312
    Keywords: nuclear pore structure ; digitonin permeabilization ; immunofluorescence ; coiled-coil proteins ; Tpr ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have identified a component of the human nuclear pore complex and have shown that it is the product of a gene involved in oncogenic activation. A monoclonal antibody raised against purified nuclear matrix proteins recognizes a single protein with an electrophoretic mobility of approximately 300 kDa and stains the nuclear envelope in a punctate pattern typical of nuclear pores. The antibody was used to screen λgt11 human cDNA libraries, and the resulting clones were sequenced and compared to sequences in the Genbank database. An exact match was found with the human tpr (for translocated promoter region) gene, a gene shown previously to be involved in the oncogenic activation of several protein kinases. Double-label immunofluorescent microscopy with the anti-Tpr antibody and an antibody to the previously characterized nuclear pore complex protein nup153 confirms that Tpr is localized to the nuclear pore complex. Tpr is located on the cytoplasmic face of the nucleus, as demonstrated by immunofluorescent staining of cells permeabilized with digitonin. Tpr is a 2,349-amino acid protein with extensive coiled-coil domains and an acidic globular C-terminus. The protein contains 10 leucine zipper motifs and numerous sites for phosphorylation by a variety of protein kinases. Immunoprecipitation of Tpr from 32P-orthophosphate-labeled cells shows that it is a phosphoprotein. Potential functions for Tpr and possible mechanisms for the transforming activity of Tpr fusion proteins are discussed. © 1996 Wiley-Liss, Inc.
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  • 163
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    Journal of Cellular Biochemistry 61 (1996), S. 127-138 
    ISSN: 0730-2312
    Keywords: β1 integrin ; β7 integrin ; α/β integrin subunit association ; VLA-4/VCAM adhesion ; integrin surface expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.
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  • 164
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    Journal of Cellular Biochemistry 61 (1996), S. 230-237 
    ISSN: 0730-2312
    Keywords: retinoic acid ; retinol ; binding ; transglutaminase ; hepatic ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: When rat liver epithelial cells were exposed to retinoic acid or retinol for 24 hr, the levels of transforming growth factor-β (TGF-β) receptors were reduced in a dose-dependent way. The decrease appeared after 12 hr of incubation with the retinoids and binding levels remained low until 24 hr after the removal of the molecules. Retinoid treatment induced a fourfold enhancement of transglutaminase (TGase) activity in the cell membranes, and cystamine, an inhibitor of TGase, prevented the decrease of the receptors. Neutralization of TGF-β by a monoclonal antibody did not suppress the decrease of the binding levels, indicating that decreased TGF-β binding capacity was not due merely to the internalization of ligand-bound receptors promoted by a stimulation of TGF-β synthesis. Thus, retinoid treatment resulted in an intense disappearance of the functional receptors from the membranes that seemed to be mediated by increased TGase activity. This phenomenon can represent a strong signal attenuation for TGF-β following retinoid exposure. © 1996 Wiley-Liss, Inc.
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  • 165
    ISSN: 0730-2312
    Keywords: human hepatoma ; dedifferentiation/progression ; PDPK ; overexpression ; kinase FA/GSK-3α ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) (a member of PDPK family) has been optimized for human hepatoma and used to demonstrate for the first time significantly increased (P 〈 0.01) activity in poorly differentiated SK-Hep-1 hepatoma (24.2 ± 2.8 units/mg) and moderately differentiated Mahlavu hepatoma (14.5 ± 2.2 units/mg) when compared to well differentiated Hep 3B hepatoma (8.0 ± 2.4 units/mg). Immunoblotting analysis revealed that increased activity of kinase FA/GSK-3α is due to overexpression of the protein. Elevated kinase FA/GSK-3α expression in human hepatoma biopsies relative to normal liver tissue was found to be even more profound. This kinase appeared to be ∼fivefold overexpressed in well differentiated hepatoma and ∼13-fold overexpressed in poorly differentiated hepatoma when compared to normal liver tissue. Taken together, the results provide initial evidence that overexpression of kinase FA/GSK-3α is involved in human hepatoma dedifferentiation/progression. Since kinase FA/GSK-3α is a PDPK, the results further support a potential role of this kinase in human liver tumorigenesis, especially in its dedifferentiation/progression. © 1996 Wiley-Liss, Inc.
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  • 166
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    Journal of Cellular Biochemistry 61 (1996), S. 246-254 
    ISSN: 0730-2312
    Keywords: marrow stromal cells ; cell morphogenesis ; attachment ; ECM ; mRNA expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Our aim was to study the role of various extracellular matrices (ECM) on growth and differentiation of marrow stromal cells in vitro. Morphology changes, gene expression, and enzymatic activities were monitored in stromal osteoblastic MBA-15 and adipocytic 14F1.1 cells. These stromal cells were plated on dishes precoated with different substrata, such as matrigel (basement membrane), collagen type I, and endothelial ECM, and compared with cells plated on protein-free dishes. Striking morphological differences were observed when the cells grew on these different substrata. Changes in cell shape and growth also led to differential mRNA expression and enzymatic activities. When MBA-15 cells were plated on collagen, there was a decrease in mRNA for alkaline phosphatase (ALK-P), osteopontin (OP), and osteonectin (ON), and an increase in mRNA for procollagen (I). A differential effect was noted on 14F1.1 cells, the mRNA for ALK-P increased, the expressions of OP and ON lowered, and no expression for procollagen (I) was monitored. MBA-15 cells cultured on matrigel had decreased mRNA for ALK-P and OP, while they had increased ON mRNA expression and remained unchanged for procollagen 1. No change in mRNA expression by 14F1.1 cells was monitored when cultured on matrigel. Functional enzymatic activities of ALK-P markedly decreased in MBA-15 cells cultured on various substrata, and increased or were unchanged in 14F1.1 cells. An additional enzyme, neutral endopeptidase (CD10/NEP), altered differentially in both cell types; this enzymatic activity increased or was unchanged when cells were cultured on these matrices. The results indicate a specific role for different ECM on various stromal cell types and their function. © 1996 Wiley-Liss, Inc.
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  • 167
    ISSN: 0730-2312
    Keywords: adhesion ; migration ; protease ; lymphocyte ; immunity ; connective tissue ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human T lymphoblastoma cells of the CD4+ 8+ Tsup-1 line, that express alpha4 and alpha5 but not alpha6 integrins of the beta1 family, and CD4+ human blood T cells bind vasoactive intestinal peptide (VIP) with high affinity, leading to increased adherence, secretion of matrix metalloproteinases (MMPs), and chemotaxis. VIP-enhanced adherence of T cells to fibronectin was inhibited significantly by neutralizing monoclonal antibodies to beta1 〉 alpha4 〉〉 alpha5, but not to alpha6. Antibodies to beta1 and alpha4 suppressed to a similarly significant extent VIP stimulation of both MMP-dependent T cell chemotaxis through fibronectin-enriched Matrigel and T cell degradation of 3H-type IV collagen in the Matrigel, without affecting VIP-evoked secretion of MMP by suspensions of T cells. The lesser inhibition of VIP-enhanced adherence of T cells to fibronectin by anti-alpha5 antibody, than antibodies to beta1 or alpha4 chains, was associated with lesser or no suppression of MMP-dependent T cell chemotaxis through Matrigel and T cell degradation of type IV collagen in the Matrigel in response to VIP. Specific beta1 integrins thus mediate interactions of stimulated T cells with basement membranes, including adherence, localized digestion by MMPs, and chemotactic passage, that promote entry of T cells into extravascular tissues. © 1996 Wiley-Liss, Inc.
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  • 168
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    Journal of Cellular Biochemistry 61 (1996), S. 459-466 
    ISSN: 0730-2312
    Keywords: adenylyl cyclase ; BAT3 ; cytoskeleton ; RAS ; signaling ; yeast ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We previously identified human CAP, a homolog of the yeast adenylyl cyclase - associated protein. Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions. We have explored the interactions of human CAP with various proteins. First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST. Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves. Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts. This interaction requires the C-terminal domain of CAP, but not the N-terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts. © 1996 Wiley-Liss, Inc.
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  • 169
    ISSN: 0730-2312
    Keywords: basic helix-loop-helix ; interleukin-1 ; interleukin-3 ; granulocyte-macrophage colony-stimulating factor ; progenitor ; transcription factor ; c-kit ligand ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The basic helix-loop-helix (bHLH) transcription factors form heterodimers and control steps in cellular differentiation. We have studied four bHLH transcription factors, SCL, lyl-1, E12/E47, and Id-1, in individual lineage-defined progenitors and hematopoietic growth factor - dependent cell lines, evaluating mRNA expression and the effects of growth factors and cell cycle phase on this expression. Single lineage-defined progenitors selected from early murine colony starts and grown under permissive conditions were analyzed by RT-PCR. SCL and E12/E47 were expressed in the vast majority of tri-, bi-, and unilineage progenitors of erythroid, macrophage, megakaryocyte, and neutrophil lineages. Expression for E12/E47 was not seen in unilineage megakaryocyte and erythroid or bilineage neutrophil/mast cell progenitors. Lyl-1 showed a more restricted pattern of expression, although expression was seen in some bi- and unilineage progenitors. No expression was detected in erythroid, erythroid-megakaryocyte-macrophage, macrophage-neutrophil, macrophage, or megakaryocytic progenitors. Id-1, an inhibitory bHLH transcription factor, was also widely expressed in all bi- and unilineage progenitors; only the trilineage erythroid-megakaryocyte-macrophage progenitors failed to show expression. Expression of these factors within a progenitor class was generally heterogeneous, with some progenitors showing expression and some not. This was seen even when two sister cells from the same colony start were analyzed. Id-1, but not E12/E47, mRNA was increased in FDC-P1 and MO7E hematopoietic cell lines after exposure to IL-3 or GM-CSF, Id-1, E12, and lyl-1 showed marked variation at different points in cell cycle in isoleucine-synchronized FDC-P1 cells. These results suggest that SCL, lyl-1, E12/E47, and Id-1 are important in hematopoietic progenitor cell regulation, and that their expression in hematopoietic cells varies in response to cytokines and/or during transit through cell cycle. © 1996 Wiley-Liss, Inc.
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  • 170
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    Journal of Cellular Biochemistry 61 (1996), S. 493-501 
    ISSN: 0730-2312
    Keywords: basement membrane ; cell binding ; epidermolysis bullosa ; extracellular matrix ; gene knock-out ; integrin ; laminin ; muscular dystrophy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Extracellular matrix molecules are often very large and made up of several independent domains, frequently with autonomous activities. Laminin is no exception. A number of globular and rod-like domains can be identified in laminin and its isoforms by sequence analysis as well as by electron microscopy. Here we present the structure-function relations in laminins by examination of their individual domains. This approach to viewing laminin is based on recent results from several laboratories. First, some mutations in laminin genes that cause disease have affected single laminin domains, and some laminin isoforms lack particular domains. These mutants and isoforms are informative with regard to the activities of the mutated and missing domains. Second, laminin-like domains have now been found in a number of other proteins, and data on these proteins may be informative in terms of structure-function relationships in laminin. Finally, a large body of data has accumulated on the structure and activities of proteolytic fragments, recombinant fragments, and synthetic peptides from laminin. The proposed activities of these domains can now be confirmed and extended by in vivo experiments. © 1996 Wiley-Liss, Inc.
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  • 171
    ISSN: 0730-2312
    Keywords: bone resorption ; tyrphostins ; genistein ; herbimycin ; osteoporosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID50s of 3 μM and 0.1 μM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 μM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of 3H-leucine, was significantly inhibited at 10 μM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 μmol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P 〈 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID50s on tyrosine kinases, with a low potential for toxicity. © 1996 Wiley-Liss, Inc.
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  • 172
    ISSN: 0730-2312
    Keywords: CoA-independent transacylase ; phospholipase D ; subcellular localization ; neutrophils ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [3H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [3H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [3H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTPγS and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [3H]alkylacyl-GP and [3H]alkylacyl-GPethanol, phospholipase D breakdown products from [3H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [3H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes. © 1996 Wiley-Liss, Inc.
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  • 173
    ISSN: 0730-2312
    Keywords: FGF ; receptors ; internalization ; photoactivable cross-linker ; heparan sulfate proteoglycans ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azido-benzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37°C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Flg receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4°C with 125I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170-180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130-160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with 125I-HSAB-FGF-2 at 4°C and then washed, were shifted to 37°C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of 125I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex. © 1996 Wiley-Liss, Inc.
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  • 174
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    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 275-289 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; HeLa S3 cells ; 2-D gel electrophoresis ; heterogeneous nuclear ribonucleoproteins ; B23 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa S3 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA. © 1996 Wiley-Liss, Inc.
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  • 175
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 314-324 
    ISSN: 0730-2312
    Keywords: M1 cell ; heme oxygenase ; transcription ; H2O2 ; TPA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: It has long been known that heme oxygenase (HO) is a key enzyme in heme catabolism and recently it was also found to acts as an oxidative stress protein to produce carbon monoxide (CO), which has similar actions to those of nitrogen monoxide (NO). Therefore, we examined transcriptional control of the HO gene in mouse M1 (myeloleukemia) cells during their differentiation into macrophages. Since the promoter region of this gene is known to have a TPA-responsive element (TRE), its expression might be regulated by a C-kinase signal transduction pathway. Then we investigated the activation of the HO gene after treatment of M1 cells with TPA and inhibitors of C-kinase. When M1 cells were treated with TPA, they differentiated into macrophage-like cells. Upon treatment with TPA, H2O2 was produced first, the nuclear proto-oncogenes fos and jun were activated, and then the HO gene was activated. The extent of transcriptional activation of the fos, jun, and HO genes in M1 cells treated with TPA was reduced by a specific inhibitor of C-kinase and a scavenger of oxygen radicals. When M1 cells were treated with H2O2 essentially the same level of transcription of the HO gene was observed, but the extent of transcriptional activation of the fos and jun genes was about half of the treatment with TPA. Super-shift assays using the TRE of the HO gene revealed that the Fos and Jun proteins from nuclei of M1 cells treated with TPA bound to the TRE, and same assays using DNA with the NF-kB motif also revealed that the active NF-kB protein from M1 cells treated with H2O2 or TPA also bound to the corresponding motif. These results strongly suggest that the HO gene in M1 cells is activated by TPA through a production of H2O2, an oxidative activation pathway of NF-kB, and a signal-transduction pathway that involves C-kinase during the differentiation of macrophages that occurs upon treatment with TPA. © 1996 Wiley-Liss, Inc.
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  • 176
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 172-180 
    ISSN: 0730-2312
    Keywords: chromatin structure ; nuclear matrix ; transcriptional activation ; replication ; recombination ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The tumor suppressor p53 is a multifunctional protein whose main duty is to preserve the integrety of the genome. This function of wild-type p53 as “guardian of the genome” is achieved at different levels, as a cell cycle checkpoint protein, halting the cell cycle upon DNA damage, and via a direct involvement in processes of DNA repair. Alternatively, p53 can induce apoptosis. Mutations in the p53 gene occur in about 50% of all human tumors and eliminate the tumor suppressor functions of p53. However, many mutant p53 proteins have not simply lost tumor suppressor functions but have gained oncogenic properties which contribute to the progression of tumor cells to a more malignant phenotype. The molecular basis for this gain of function of mutant p53 is still unknown. However, mutant (mut) p53 specifically binds to nuclear matrix attachment region (MAR) DNA elements. MAR elements constitute important higher order regulatory elements of chromatin structure and function. By binding to these elements, mut p53 could modulate important cellular processes, like gene expression, replication, and recombination, resulting in phenotypic alterations of the tumor cells. Mut p53 thus could be the first representative of a new class of oncogenes, which exert their functions via long-range alterations or perturbation of chromatin structure and function. © 1996 Wiley-Liss, Inc.
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  • 177
    ISSN: 0730-2312
    Keywords: dexamethasone ; actin ; polymerization ; Ishikawa cells ; cAMP ; actinomycin D ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Glucocorticoids, in addition to their well characterized effects on the genome, may affect cell function in a manner not involving genomic pathways. The mechanisms by which the latter is achieved are not yet clear. A possible means for this action may involve the actin cytoskeleton, since the dynamic equilibrium of actin polymerization changes rapidly following exposure to several stimuli, including hormones. The aim of the present work was to find out if glucocorticoids exert rapid, nongenomic effects on actin polymerization in Ishikawa human endometrial cells, which represent a well characterized in vitro cell model expressing functional glucocorticoid receptors. Short term exposure of the cells to the synthetic glucocorticoid dexamethasone resulted in an overall decrease of the G/total-actin ratio in a time- and dose-dependent manner. Specifically, in untreated Ishikawa cells the G/total-actin ratio was 0.48 ± 0.01 (n = 26). It became 0.35 ± 0.01 (n = 13, P 〈 0.01) following exposure to 10-7 M dexamethasone for 15 min. This was induced by a significant decrease of the cellular G-actin level, without affecting the total actin content, indicating a rapid actin polymerization. This conclusion was fully confirmed by direct fluorimetry measurements, that showed a significant increase of the F-actin content by 44% (n = 6, P 〈 0.001) in cells treated with dexamethasone (10-7 M, 15 min). The rapid dexamethasone-induced alterations of the state of actin polymerization were further supported by fluorescence microscopy. The latter studies showed that the microfilaments of cells pretreated with 10-7 M dexamethasone for 15 min were more resistant to various concentrations of the antimicrofilament drug cytochalasin B, compared to untreated cells, implying microfilament stabilization. The action of dexamethasone on actin polymerization seems to be mediated via specific glucocorticoid binding sites, since the addition of the glucocorticoid antagonist RU486 completely abolished its effect. Moreover, it appears to act via non-transcriptional pathways, since actinomycin D did not block the dexamethasone-induced actin polymerization. In addition, cell treatment with 10-7 M dexamethasone for 15 min fully reversed the forskolin-, but not the 8-bromo-cAMP-induced actin depolymerization. In line with these findings, the cAMP content of Ishikawa cells was decreased by 29.2% after a 15 min treatment with 10-7 M dexamethasone (n = 4, P 〈 0.01). In conclusion, our results showed that dexamethasone induces rapid, time-, and dose-dependent changes in actin polymerization dynamics in Ishikawa cells. This action seems to be mediated via cAMP, involving probably nongenomic pathways. The above findings offer new perspectives for the understanding of the early cellular responses to glucocorticoids. © 1996 Wiley-Liss, Inc.
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  • 178
    ISSN: 0730-2312
    Keywords: monocyte chemoattractant protein-1 ; gene expression ; pig artery ; balloon injury ; monocyte/macrophages ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are potent chemokines which attract circulating monocytes and neutrophils respectively to inflamed tissues. JE/MCP-1 gene expression has been previously studied in rabbit aortae after endothelial denudation and the rapid appearance of this transcript was thought to precede emigration of phagocytes. We now report MCP-1 gene expression following de-endothelialization of iliac arteries in the pig, a species which can develop spontaneous atherosclerosis. Using Northern blot analysis, we demonstrated that MCP-1 mRNA was rapidly induced in pig arteries at 2 h and continued to increase to reach a maximum at 8 h before returning to low levels at 16-24 h after injury. The increase seen for MCP-1 mRNA at 8 h was also observed for IL-8 mRNA but was not apparent for growth-related gene expressions, urokinase-type plasminogen activator (u-PA), and plasminogen activator inhibitor-1 (PAI-1). Since smooth muscle cells, endothelial cells, and phagocytes are all capable of expressing MCP-1, we examined pig arteries for immunostaining using a monoclonal antibody to human MCP-1 (5D3-F7). At 8 h after injury, the predominant cell type staining positive for MCP-1 was the monocyte/macrophage. Staining was also observed in occasional scattered neutrophils, but MCP-1 protein could not be detected in smooth muscle cells or on extracellular matrix within the sensitivity constraints posed by our methodology. Our results are consistent with invading monocyte/macrophages having a major input into the production of this chemokine in the arterial wall following injury. The fact that MCP-1 expression accompanied monocyte/macrophage presence in damaged artery, rather than preceding it, is suggestive that continued MCP-1 expression is required for functions other than chemoattraction. © 1996 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 179
    ISSN: 0730-2312
    Keywords: GLRP ; T-lymphocyte ; immune response ; central nervous system ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Coordination of the immune response to injury or disease in the brain is postulated to involve bi-directional discourse between the immune system and the central nervous system. This cross communication involves soluble mediators, including various growth factors, cytokines, and neuropeptides. In this report, we demonstrate that the supernatant from activated T-lymphocytes is able to induce the transcription of a potent cytokine, TGF-β2 in glial cells. The activating stimulus invokes signaling mechanisms distinct from known kinase or protease pathways. Activation of TGF-β2 transcription correlates with the loss of binding activity for an 80 kDa glial labile repressor protein, GLRP, to a responsive region within the TGF-β2 promoter. Although GLRP shares some characteristics with the inducible transcription factor AP-1, it appears to be distinct from known AP-1 family members. These data along with previous observations demonstrating the potent immunosuppressive activity of TGF-β2, support a model for a feedback mechanism between the activated T-lymphocytes and astrocytes via TGF-β2 to regulate the immune response. © 1996 Wiley-Liss, Inc.
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  • 180
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 454-466 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; histone H5 ; transcription ; transcription factors ; erythroid development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear matrix has roles in organizing nuclear DNA and in controlling transcription. Transcription factors are associated with the nuclear matrix, with the spectra of transcription factors differing from one cell type to another. In this study we identified the transcription factors and enzymes functioning in the regulation of gene expression that were associated with nuclear matrix and nonmatrix nuclear fractions in erythrocytes isolated from chick embryos at different stages of development, anemic and normal adult birds. We found that the primitive erythroid nuclear matrix had the greatest histone deacetylase activity and highest levels of several transcription factors, including GATA-1, CACCC-binding proteins, and NF1. These transcription factors have key roles in erythroid-specific gene expression. The levels of these transcription factors were lower in the nonmatrix and matrix fractions isolated from definitive erythrocytes. For primitive and definitive erythrocytes, the level of CACCC-binding proteins in the nuclear matrix fraction was greater than that of Sp1. The relative levels of these transcription factors were reversed in the nonmatrix fraction. Casein kinase II was not found in erythroid nuclear matrices. The observed erythroid lineage specific alterations in erythroid nuclear matrix transcription factor composition and abundance may be involved in erythroid-specific gene expression. © 1996 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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  • 181
    ISSN: 0730-2312
    Keywords: adhesion ; breast cancer cells ; thrombospondin ; receptors ; proteoglycans ; heparin-binding peptides ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Thrombospondin is an adhesive glycoprotein that promotes breast cancer cell adhesion to human vascular endothelial cells (Incardona et al., 1995). In this study, we have identified the molecular domains of thrombospondin that mediate its binding to specific receptors on the human breast adenocarcinoma cell line, MDA-MB-231. Two recombinant fragments from the amino-terminus (TSPN18 and TSPN28), and the fusion proteins of the type 1 and type 2 repeats of human thrombospondin, inhibited binding of radiolabeled thrombospondin to MDA-MB-231 cells in suspension by 40-60% at 50 μg/ml whereas the type 3 repeat, carboxy-terminus and unfused glutathione-S-transferase as well as the synthetic peptide Gly-Arg-Gly-Asp-Ser (500 μg/ml) had little or no effect. Herapin and various glycosaminoglycans as heparan sulfate, chondroitin sulfates A, B or C, and fucoidan inhibited thrombospondin binding to MDA-MB-231 cells by more than 60% whereas dextran sulfate had only little effect. Treatment of cells with heparitinase, chondroitinase ABC, and hyaluronidase, but not with neuraminidase, induced 30-50% inhibition of thrombospondin binding suggesting the participation of both heparan sulfate and chondroitin sulfate cell surface-associated molecules. Inhibition of proteoglycan sulfation by chlorate or inhibition of glycosaminoglycan chain formation by two β-D-xylosides also led to a substantial inhibition of thrombospondin binding. Our results indicate that several domains within the thrombospondin molecule, namely the amino-terminus, type 1 and type 2 repeats, participate in its binding to specific receptors bearing sulfated glycosaminoglycans on MDA-MB-231 cells. Biological assays have indicated that, in addition to these domains, the peptide Gly-Arg-Gly-Asp-Ser inhibited MDA-MB-231 cell attachment to thrombospondin suggesting that the last type 3 repeat of the molecule may also contribute to its cell adhesive activity. © 1996 Wiley-Liss, Inc.
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  • 182
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 506-515 
    ISSN: 0730-2312
    Keywords: heat shock ; pre-rRNA processing ; S-100 extract ; U3 snoRNA ; 3′ processing ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effect of heat shock on pre-rRNA processing at the primary site within external transcribed spacer region 1 (ETS1) was studied in S-100 extract derived from mouse lymphosarcoma cells. In vivo labeling with [32P]orthophosphate showed that the synthesis of the rRNA precursor and its processing to 28S and 18S rRNAs were inhibited significantly due to heat shock. The processing activity was reduced by 50% at 1 h and was completely blocked following 2-h exposure of cells at 42°C. Mixing S-100 extracts from the control and heat-treated cells did not affect the processing activity in the control extract, which proves the absence of a nuclease or other inhibitor(s) of processing in the extract from the heat-shocked cells. Heat shock did not affect interaction between pre-rRNA and U3 snoRNA, a prerequisite for the processing at the primary site, but significantly altered RNA-protein interaction. Three polypeptides of 200, 110, 52 kDa that specifically cross-link to pre-rRNA spanning the primary processing site were inactivated after heat shock. Hyperthermia did not alter 3′ end processing of SV40L pre-mRNA. © 1996 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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  • 183
    ISSN: 0730-2312
    Keywords: osteosarcoma ; chondrosarcoma ; GCT ; oncogene alterations ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated the structure and the expression of various oncogenes in three of the most common human bone tumors - osteosarcoma (36 samples from 34 patients), giant cell tumor (10 patients), and chondrosarcoma (18 patients) - in an attempt to identify the genetic alterations associated with these malignancies. Alterations of RB and p53 were detected only in osteosarcomas. Alterations of c-myc, N-myc, and c-fos were detected in osteosarcomas and giant cell tumors. Ras alterations (H-ras, Ki-ras, N-ras) were rare. Chondrosarcomas did not contain any detectable genetic alterations. Our results suggest that alterations of c-myc, N-myc, and c-fos oncogenes occur in osteosarcomas, in addition to those previously described for the tumor suppressor genes RB and p53. Moreover, statistical analyses indicate that c-fos alterations occur more frequently in osteosarcoma patients with recurrent or metastatic disease. © 1996 Wiley-Liss, Inc.
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  • 184
    ISSN: 0730-2312
    Keywords: Src kinase ; mercuric chloride ; redox ; sulfhydryl group ; receptor polymerization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Little is known about the regulatory mechanism of c-Src kinase in cells except the suggested regulation through phosphorylation and dephosphorylation of its carboxyl terminal tyrosine residue (Y527). We here demonstrated that exposure of NIH3T3 cells to mercuric chloride (HgCl2) induces both aggregation and activation of Src kinase protein through a redox-linked mechanism. The aggregation of Src proteins was suggested to be induced by the sulfhydryl groups-to-Hg2+ reaction-mediated polymerization of cell membrane proteins to which the Src proteins associate noncovalently. The possibility was ruled out that the aggregation occurred secondarily to the promotion of protein tyrosine phosphorylation. Further study revealed that the Src kinase was activated by HgCl2 at least in part independent of the known Csk kinase-linked or Y527-phosphorylation/dephosphorylation-mediated control. Correspondingly, CNBr cleavage mapping of phosphopeptides for autophosphorylated c-Src protein demonstrated selective promotion of phosphorylation at Y416 in HgCl2-treated cells without obvious change in the phosphorylation level at Y527. These results suggest a unique protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase in NIH3T3 cells. © 1996 Wiley-Liss, Inc.
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  • 185
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 162-173 
    ISSN: 0730-2312
    Keywords: Topo IIα ; Topo IIβ ; interphase ; mitosis ; mitogenic stimulation ; nucleoplasm ; nucleolus ; lymphocytes ; HeLa ; immunofluorescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have carried out immunofluorescence labelling of two human cell types, HeLa cells and peripheral blood lymphocytes, prepared by several different fixation/permeabilization protocols using a variety of antibodies against DNA Topoisomerase II (Topo II). We have found that the distribution of Topo IIα was overall similar during interphase and mitosis to that previously reported, regardless of antibody and of sample preparation. On the other hand, the interphase distribution of Topo IIβ was quite variable, depending both on the antibody and on the method used to prepare the sample. Our interpretation of the data is that, like Topo IIα, Topo IIβ is primarily a nucleoplasmic protein, but that unlike Topo IIα, small amounts are also associated with intranucleolar chromatin. © 1996 Wiley-Liss, Inc.
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  • 186
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 185-198 
    ISSN: 0730-2312
    Keywords: extracellular matrix ; remodeling ; collagenase ; collagen ; dilated cardiomyopathy ; congestive heart disease ; end-stage heart failure ; matrix metalloproteinase ; tissue inhibitor of metalloproteinase ; differential display mRNA analysis ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Extracellular matrix metalloproteinases (MMPs) are activated in dilated cardiomyopathic (DCM) hearts [Tyagi et al. (1996): Mol Cell Biochem 155:13-21]. To examine whether the MMP activation is occurring at the gene expression level, we performed differential display mRNA analysis on tissue from six dilated cardiomyopathy (DCM) explanted and five normal human hearts. Specifically, we identified three genes to be induced and several other genes to be repressed following DCM. Southern blot analysis of isolated cDNA using a collagenase cDNA probe indicated that one of the genes induced during DCM was interstitial collagenase (MMP-1). Northern blot analysis using MMP-1 cDNA probe indicated that MMP-1 was induced three- to fourfold in the DCM heart as compared to normal tissue. To analyze posttranslational expression of MMP and tissue inhibitor of matrix metalloproteinase (TIMP) we performed immunoblot, immunoassay, and substrate zymographic assays. TIMP-1 and MMP-1 levels were 37 ± 8 ng/mg and 9 ± 2 ng/mg in normal tissue specimens (P 〈 0.01) and 2 ± 1 ng/mg and 45 ± 11 ng/mg in DCM tissue (P 〈 0.01), respectively. Zymographic analysis demonstrated lytic bands at 66 kDa and 54 kDa in DCM tissue as compared to one band at 66 kDa in normal tissue. Incubation of zymographic gel with metal chelator (phenanthroline) abolished both bands suggesting activation of neutral MMP in DCM heart tissue. TIMP-1 was repressed approximately twentyfold in DCM hearts when compared with normal heart tissue. In situ immunolabeling of MMP-1 indicated phenotypic differences in the fibroblast cells isolated from the DCM heart as compared to normal heart. These results suggest disruption in the balance of myopathic-fibroblast cell ECM-proteinase and antiproteinase in ECM remodeling which is followed by dilated cardiomyopathy. © 1996 Wiley-Liss, Inc.
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  • 187
    ISSN: 0730-2312
    Keywords: regulation of transcription ; control of proliferation ; vitamin D3 analogues ; vitamin D3 receptor ; limited protease digestion assay ; lymphocytes ; breast cancer cells ; promoter selectivity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The biological active form of vitamin D3, 1,25-dihydroxyvitamin D3 (VD), regulates cellular growth and differentiation. This provides the hormone with an interesting therapeutic potential. However, hypercalcemia is a side effect, which is caused by VD's classical action, the regulation of calcium homeostasis. This made the need for VD analogues with selectively increased cell regulatory properties. Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD. In this report the biological properties of the compounds ZK161422 and ZK157202, which are 20-methyl- and 20-methyl-23-eneanalogues, respectively, have been analyzed in comparison with VD. Both compounds show about 2-fold lower affinity to the VD receptor (VDR) than VD. However, compared to VD, their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells. Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold, ZK161422 has the same calcium-mobilizing potency as VD. Moreover, ZK161422, but not ZK157202, showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides (IP9) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides (DR3). This observation supports a model, in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues. © 1996 Wiley-Liss, Inc.
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  • 188
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    Journal of Cellular Biochemistry 63 (1996), S. 239-251 
    ISSN: 0730-2312
    Keywords: lymphocyte activation ; Krebs cycle ; energy metabolism ; immunosuppressives ; cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Rapamycin (RAPA) strongly inhibits lymphocyte activation and proliferation, but does not affect most of the activation-related gene expression at the mRNA level. In order to understand the mechanism of action of RAPA and to gain further insights in lymphocyte signalling which is impaired by RAPA, we screened for RAPA-sensitive genes using differential hybridization. The expression of human aldolase A gene was found to be inducible during T and B cell activation, and the induction was repressed by RAPA at both the mRNA and enzymatic levels. The other two important immunosuppressants, cyclosporin A and FK506, also inhibited the mitogen-induced upregulation. However, none of these three drugs inhibited the constitutive expression. There was no fluctuation of aldolase A expression during the cell cycle, and RAPA failed to block the first cell cycle after synchronization in Jurkat cells. However, the second cycle was hampered by RAPA, and this was correlated with the inhibition of aldolase A expression during this later stage. Since aldolase A is a key enzyme in glycolysis and lymphocytes mainly depend on glycolysis for energy supply, the data from this study suggest that aldolase A might be one of the downstream targets of RAPA. The inhibition of the enzyme upregulation might deprive the cells of additional supply of energy, and prevent the cells from entering an optimal status for proliferation. © 1996 Wiley-Liss, Inc.
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  • 189
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    Journal of Cellular Biochemistry 63 (1996), S. 268-279 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; mitosis ; Drosophila embryo ; monoclonal antibody ; spindle formation ; nucleus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Little is known about what determines the nuclear matrix or how its reorganization is regulated during mitosis. In this study we report on a monoclonal antibody, mAb2A, which identifies a novel nuclear structure in Drosophila embryos which forms a diffuse meshwork at interphase but which undergoes a striking reorganization into a spindle-like structure during pro- and metaphase. Double labelings with α-tubulin and mAb2A antibodies demonstrate that the microtubules of the mitotic apparatus co-localize with this mAb2A labeled structure during metaphase, suggesting it may serve a role in microtubule spindle assembly and/or function during nuclear division. That the mAb2A-labeled nuclear structure is essential for cell division and/or maintenance of nuclear integrity was directly demonstrated by microinjection of mAb2A into early syncytial embryos which resulted in a disintegration of nuclear morphology and perturbation of mitosis. © 1996 Wiley-Liss, Inc.
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  • 190
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    Journal of Cellular Biochemistry 63 (1996), S. 311-319 
    ISSN: 0730-2312
    Keywords: protein phosphatase 2A ; endothelial cells ; cyclic strain ; proliferation ; okadaic acid ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic strain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C. © 1996 Wiley-Liss, Inc.
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  • 191
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    Journal of Cellular Biochemistry 63 (1996), S. 366-373 
    ISSN: 0730-2312
    Keywords: capsule ; lipid droplet ; Leydig cell ; monoclonal antibody ; immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In this report, we provide direct evidence for the presence of a lipid droplet-associated capsule in hamster steroidogenic Leydig cells by using a monoclonal antibody A2. Leydig cells are characterized by containing many lipid droplets and having 3β-hydroxysteroid dehydrogenase activity. Immunofluorescence staining with this antibody demonstrated a rim or capsule surrounding the lipid droplets in Leydig cells, a pattern not seen with anti-vimentin antibody. Immunogold labelling confirmed ultrastructurally that antibody binding was distributed on the lipid droplet surface. In order to investigate the possible function of the capsule, we examined the morphological changes induced in the capsule following stimulation with LH or dibutyryl cAMP; the fluorescent intensity of the capsule was seen to gradually decrease, accompanied by a decrease in number and size of lipid droplets, and the response to both reagents was time- and concentration-dependent. We thus conclude that hormonal stimulation resulting in the detachment of certain capsular proteins from the surface of lipid droplets is mediated via the cAMP signaling pathway and may allow cholesterol ester hydrolytic enzyme direct access to its substrate in the lipid droplet. © 1996 Wiley-Liss, Inc.
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  • 192
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    Journal of Cellular Biochemistry 63 (1996), S. 453-462 
    ISSN: 0730-2312
    Keywords: FBPase ; gluconeogenesis ; perinuclear association ; metabolic zonation ; immunolocalization ; subcellular fractionation ; confocal microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The localization of fructose 1,6-bisphosphatase (D-Fru-1,6-P2-1-phosphohydrolase, EC 3.1.3.11) in rat kidney and liver was determined immunohistochemically using a polyclonal antibody raised against the enzyme purified from pig kidney. The immunohistochemical analysis revealed that the bisphosphatase was preferentially localized in hepatocytes of the periportal region of the liver and was absent from the perivenous region. Fructose-1,6-bisphosphatase was also preferentially localized in the cortex of the kidney proximal tubules and was absent in the glomeruli, loops of Henle, collecting and distal tubules, and in the renal medulla. As indicated by immunocytochemistry using light microscopy and confirmed with the use of reflection confocal microscopy, the enzyme was preferentially localized in a perinuclear position in the liver and the renal cells. Subcellular fractionation studies followed by enzyme activity assays revealed that a majority of the cellular fructose-1,6-bisphosphatase activity was associated to subcellular particulate structures. Overall, the data support the concept of metabolic zonation in liver as well as in kidney, and establish the concept that the Fructose-1,6-bisphosphatase is a particulate enzyme that can not be considered a soluble enzyme in the classical sense. © 1996 Wiley-Liss, Inc.
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  • 193
    ISSN: 0730-2312
    Keywords: transcription initiation ; CpG island ; transcription factor AP2 ; transcription factor Sp1 ; osteoblasts ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transforming growth factor (TGF-β) binds several discrete membrane proteins. Of these, a type I receptor appears indispensable for signal transduction. Previous examination of TGF-β receptor expression has been limited to changes in cell surface protein, and more recently, mRNA abundance. In order to learn more about TGF-β function and receptor expression during osteogenesis, we have now cloned a 4 kilobase (kb) DNA fragment 5' proximal to the coding region of the rat TGF-β type I receptor gene. Sequence analysis revealed multiple elements compatible with transcription initiation, including a properly positioned and oriented CCAAT box, six Sp1 binding sites (three defining GC boxes), and two strong AP2 binding sites within a 0.7 kb span directly upstream of the coding region. The 3' terminal 0.3 kb span comprises a GC-enriched (77%) so-called CpG island that, like other similarly organized promoters, lacks a TATA box. Primer extension and RNase protection studies with cRNAs from this area show multiple initiation sites within 220 bp 5' proximal to the initial methionine codon. Transient transfections using nested, deleted, and inverted promoter sequences demonstrated maximal reporter expression by a 1 kb fragment encompassing all of these elements. Truncation of the 1 kb fragment from the 5' and 3' ends indicated the need for several elements for peak promoter activity. These results, and transfections in fetal rat bone and dermal cells, suggest that this promoter contains elements that specify basal and conditional expression of the TGF-β type I receptor in bone. © 1996 Wiley-Liss, Inc.
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  • 194
    ISSN: 0730-2312
    Keywords: protein kinase C ; Drosophila melanogaster ; embryonic neurons ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Embryonic neurons were cultured from transgenic Drosophila melanogaster expressing a highly specific pseudosubstrate inhibitor of protein kinase C (PKC). Flies homozygous for this transgene, which is under the control of the yeast UAS promoter, were crossed to flies homozygous for the yeast heat shock inducible transcription factor GAL 4. Following heat shock, the progeny express the pseudosubstrate inhibitor at high levels. This strategy, which has the advantage of avoiding the non-specific effects of drugs, was used to study the role of PKC in process growth of cultured, differentiating neuroblasts. An external gold particle labeling procedure using a cell surface antigen expressed by mature neurons and processes was used to visualize neuronal processes directly in the scanning electron microscope. We observed that cell cultures expressing a low concentration of the pseudosubstrate inhibitor showed a significant decrease in the number of type I and II processes as compared to control cultures, while the proportions of neuroblasts, ganglion mother cells (GMCs), and mature neurons in the clusters were little affected. © 1996 Wiley-Liss, Inc.
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  • 195
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    Journal of Cellular Biochemistry 61 (1996), S. 18-25 
    ISSN: 0730-2312
    Keywords: osteoblasts ; calvaria ; invasion ; prostate ; PC-3 cells ; differentiation ; metastasis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone metastasis is a common event and a major cause of morbidity in prostate cancer patients. After colonization of bone, prostate cells induce an osteoblastic reaction which is not associated with marrow fibrosis (i.e., osteoblast but not fibroblast proliferation). In the present study we test the hypothesis that the tumoral prostatic cell line (PC-3) secretes factors that block the osteoblast differentiation process, resulting in an increase of the relative size of the proliferative cell pool. Our results, using fetal rat calvaria cells in culture, show that conditioned medium from PC-3 cells (PC-3 CM) stimulates osteoblast proliferation and inhibits both alkaline phosphatase (AP) activity (an early differentiation marker) and the mineralization process, measured as calcium accumulation (late differentiation marker). The inhibition of the expression of AP and mineralization depends on the presence of PC-3 CM during the proliferative phase of culture and suggests that both processes occur in a nonsimultaneous fashion. The inhibitory effect of PC-3 CM was not reverted by dexamethasone, which would indicate that prostatic-derived factors and the glucocorticoid do not share a common site of action. Measurement of the proliferative capacity of subcultures from control and treated cells demonstrates that PC-3 CM treatment induces the maintenance of the proliferative potential that characterizes undifferentiated precursor cells. © 1996 Wiley-Liss, Inc.
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  • 196
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    Journal of Cellular Biochemistry 61 (1996), S. 97-108 
    ISSN: 0730-2312
    Keywords: aggregin ; chemical modification ; ADP-induced platelet responses ; NBD-Cl ; cAMP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: ADP-induced platelet responses play an important role in the maintenance of hemostasis. There has been disagreement concerning the identity of an ADP receptor on the platelet surface. The chemical structure of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) shows considerable resemblance to that of the adenine moiety of adenine-based nucleotides. The reagent has been previously used by other investigators as an affinity label for adenine nucleotide-requiring enzymes, such as mitochondrial ATPase and the catalytic subunit of cAMP-dependent protein kinase. Since ADP-induced platelet responses depend on the binding of ADP to its receptor, we investigated the effect on ADP-induced platelet responses and the nature of ADP-binding protein modified by NBD-Cl. NBD-Cl inhibited ADP-induced shape change and aggregation of platelets in platelet-rich plasma in a concentration- and time-dependent manner. NBD-Cl also inhibited ADP-induced shape change, aggregation, exposure of fibrinogen binding sites, secretion, and calcium mobilization in washed platelets. NBD-Cl did not act as an agonist for platelet shape change and aggregation. Covalent modification of platelets by NBD-Cl blocked the ability of ADP to antagonize the increase in intracellular levels of cAMP mediated by iloprost (a stable analogue of prostaglandin I2). NBD-Cl was quite specific in inhibiting platelet aggregation by those agonists, e.g., ADP, collagen, and U44619 (a thromboxane mimetic), that completely or partially depend on the binding of ADP to its receptor. Autoradiogram of the gel obtained by SDS-PAGE of solubilized platelets modified by [14C]-NBD-Cl showed the presence of a predominant radiolabeled protein band at 100 kDa corresponding to aggregin, a putative ADP receptor. The intensity of this band was considerably decreased when platelets were either preincubated with ADP and ATP or covalently modified by a sulfhydryl group modifying reagent before modification by [14C]-NBD-Cl. These results (1) indicate that covalent modification of aggregin by NBD-Cl contributed to loss of the ADP-induced platelet responses, and (2) suggest that there is a sulfhydryl group in the ADP-binding domain of aggregin. © 1996 Wiley-Liss, Inc.
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  • 197
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    Journal of Cellular Biochemistry 63 (1996), S. 108-111 
    ISSN: 0730-2312
    Keywords: duct carcinoma in situ ; nuclear grade necrosis ; prognostic features ; local recurrence ; invasive transformation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In the last 6 years a number of non-randomized, predominantly single institutional trials of breast conservation therapy (BCT) with DCIS, have demonstrated that it constitutes a very heterogeneous group of diseases with markedly different risks of local recurrence and invasive transformation. There has been a consensus that DCIS, which exhibits a “comedo” morphology, generally defines a high risk group. Most studies, moreover, have identified the same two features, nuclear grade and necrosis, as contributing most significantly to prognosis [4-6]. Nuclear grade and necrosis have been identified as independent prognostic variables in several studies [5,6]. High nuclear grade DCIS which exhibits comedo necrosis defines the majority of all DCIS which will result in local recurrence and invasive transformation after BCT.Studies utilizing image cytometry, to determine ploidy and S-phase fraction and immunohistochemical studies of proliferation and oncogene distribution have shown a significant association with morphologically identified high nuclear grade and aneuploidy, high S-phase fraction or proliferation rate, presence of HER-2/neu and P53 oncogenes and absence of estrogen receptors. Generally the inverse of this association is seen with low nuclear grade DCIS. However, initial hopes that these adjunctive studies would identify subsets within the high nuclear grade group which might be more likely to recur have not been fulfilled. J. Cell. Biochem. 25S:108-111. © 1997 Wiley-Liss, Inc.
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  • 198
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    Journal of Cellular Biochemistry 63 (1996), S. 123-130 
    ISSN: 0730-2312
    Keywords: carcinogenesis ; predisposing mutation ; malignancy ; DNA testing ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Identification of cohorts at genetic risk for cancer offers unique research opportunities to explore the steps in carcinogenesis, from the inheritance of a predisposing mutation to the development of preinvasive lesions or overt malignancy, and to evaluate interventions to modulate the carcinogenic process. However, cancer prevention strategies for most inherited cancer predisposition syndromes are of unproven benefit, and the potential for adverse psychosocial effects and employment or insurance discrimination associated with genetic testing is substantial. Thus testing for genetic cancer risk remains highly controversial, and the National Center for Human Genome Research and the American Society of Human Genetics advise DNA testing for presymptomatic identification of cancer risk only in the setting of a carefully monitored research environment.The commercial availability of predictive genetic testing, particularly for inherited susceptibility to cancer, has focused attention not only on the urgent need for research in cancer prevention for cohorts at genetic cancer risk but also on ethical considerations surrounding clinical prevention research in genetic risk groups. This paper addresses the interrelationship of ethical and scientific issues in conducting chemoprevention research in these cohorts, especially for those studies which require presymptomatic testing for specific gene mutations as a study entry criterion or as a criterion for stratification. Practical approaches to study design and implementation issues for chemoprevention research in genetic risk cohorts are discussed, emphasizing the interactions of ethical and scientific considerations at all levels of the research process. J. Cell. Biochem. 25S:123-130. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
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  • 199
    ISSN: 0730-2312
    Keywords: acquired risk ; chemoprevention ; colon ; genetic risk ; neoplasia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The progressively abnormal development of epithelial cells prior to tumor development leads to widely differing chemopreventive approaches. The diversity of these approaches has resulted in different assays to measure the activities of the agents. To apply these assays to preclinical studies, we have developed rodent models in which different stages of evolution of colonic neoplasia are expressed. In one model mice carrying a truncated Apc allele with a nonsense mutation in exon 15 have been generated by gene targeting and embryonic stem cell technology (Apc1638 mice). These mice develop multiple gastrointestinal lesions including adenomas and carcinomas, focal areas of high grade dysplasia (FAD) and polypoid hyperplasias with FADS.The incidence of inherited colonic neoplasms has now been modulated by a chemopreventive regimen. Colonic lesions significantly increased in Apc1638 mice on a Western-style diet, compared to Apc1638 mice on AIN-76A diet which has lower fat content and higher calcium and vitamin D. These studies have also been carried out in normal mice, and have demonstrated without any chemical carcinogen that a Western-style diet induced colonic tumorigenesis. Modulation of cell proliferation has also been induced by Western-style diets in other organs including mammary gland, pancreas and prostate. These findings are leading to the development of new preclinical models for evaluating the efficacy of many classes of chemopreventive agents. J. Cell. Biochem. 25S:136-141. © 1997 Wiley-Liss, Inc.
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  • 200
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    Journal of Cellular Biochemistry 63 (1996), S. 156-164 
    ISSN: 0730-2312
    Keywords: carcinogenesis ; chemoprevention ; prostate cancer ; prostatic intraepithelial neoplasia ; prostatic neoplasms ; surrogate endpoint biomarkers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The most efficient strategy for chemoprevention clinical trials are short-term studies which focus on surrogate endpoint biomarkers (SEBs) in high-risk target populations. High-grade prostatic intraepithelial neoplasia (PIN) is the most likely precursor of prostate cancer, and is found in a significant number of routine contemporary needle biopsies without cancer. The frequency and extent of PIN are decreased with androgen deprivation therapy, suggesting that it is a suitable endpoint biomarker for modulation. Potential SEBs for screening chemopreventive agents for prostate cancer in short-term Phase II trials include (1) histologic premalignant lesions, such as high-grade PIN; (2) biochemical markers, including prostate-specific antigen (PSA) serum concentration; and (3) morphometric markers, including nuclear texture, shape, and roundness; size and number of nucleoli; and number of apoptotic bodies; (4) proliferation markers, including MIB-1 and PCNA; (5) genetic markers, including nuclear DNA content (ploidy), oncogene c-erbB-2 (HER-2/neu) expression, fluorescence in situ hybridization for chromosome 8; and PSA-producing cells in the blood detected by reverse transcriptase polymerase chain reaction; and (6) differentiation markers, such as microvessel density as a determinant of angiogenesis. Each of these endpoint biomarkers is measured easily and accurately in serum or in tissue specimens such as formalin-fixed, paraffin-embedded needle biopsies, and may be modifiable by intervention. The clinical utility of these biomarkers as modulatable endpoints in prostate cancer chemoprevention needs to be demonstrated in future clinical trials. J. Cell. Biochem. 25S:156-164. © 1997 Wiley-Liss, Inc.
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