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  • 101
    Electronic Resource
    Electronic Resource
    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 229-240 
    ISSN: 1069-8299
    Keywords: filling of thin section ; finite element method ; surface tension ; interface element ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: An interface element to model the pressure discontinuity due to surface tension when applied to the filling of a thin section cavity is presented. The equations used to form the element matrix for the interface element are the line integral form of the continuity and momentum equations. During the development of the finite element model, the pressure difference across the free surface due to surface tension is treated as an additional traction and is applied to all element sides which form the free surface. Simple numerical examples are then presented to illustrate the technique on the filling of a rectangular thin section cavity. © 1998 John Wiley & Sons, Ltd.
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  • 102
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 253-269 
    ISSN: 1069-8299
    Keywords: potential flow ; optimization approach ; sensitivity analysis ; adjoint variable method ; finite elements ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: Potential flow problems around immersed bodies have been treated by an optimization approach. When the stream function is used as the field variable, the boundary values may not be known a priori and may be taken as the decision parameters to minimize integral objective functionals. The circulation integrals around the immersed bodies or the Kutta condition at the trailing edges of the bodies may be used to construct the objective function of optimization. The sensitivity analysis needed for the minimization process is performed by the adjoint variable method, while the numerical solutions of the primary (flow) and adjoint equations have been obtained by the finite element method. Having checked the present method with exact solutions and the classical superposition method, several flow problems involving one or more immersed bodies with or without circulation are investigated numerically. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 12 Ill.
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  • 103
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 289-304 
    ISSN: 1069-8299
    Keywords: stress intensity factors (SIF) ; finite element method (FEM) ; reciprocal work contour integral (RWCI) ; path-independent integrals (PII) ; displacement correlation technique (DCT) ; quarter-point displacement technique (QPDT) ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A new method for improving the approximations of stress intensity factors computed from path-independent integrals is developed. The method uses Richardson's extrapolation. Numerical results are given to show the efficiency and the stability of the present method. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 11 Ill.
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  • 104
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 321-333 
    ISSN: 1069-8299
    Keywords: eigenvalue analysis ; sensitivity evaluation ; large-scale systems ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper presents a general rank-1 matrix formula which allows for proper rearrangement of individual terms in multiproduct forms involving vectors and matrices. A far-reaching application of the new matrix formula to eigenvalue sensitivity evaluation is presented in the paper. Such an application reduces the sensitivity expressions to elegant, very fast and recursive forms with substantial savings in computer resources. The formula is applicable to rank-1 matrices of special structures which may constitute derivatives of the system state matrix, which is widely used in control system applications, with respect to various parameters of interest. In such cases, the use of the rank-1 formula yields exact non-approximate solutions which are identical to those obtained by other conventional formulas. The applicability of the rank-1 formula is believed to cover a wide variety of practical engineering systems pertaining to control and stability. © 1998 John Wiley & Sons, Ltd.
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  • 105
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 719-730 
    ISSN: 1069-8299
    Keywords: phase-change problems ; conduction-advection equation ; upwind weight function ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A finite element formulation for solving transient multidimensional phase-change problems considering advective effects is presented. This temperature-based formulation includes the definition of a phase-change function able to deal with classical isothermal and non-isothermal phase-change cases. Moreover, a new upwind weight function is defined in order to avoid numerical oscillations in problems with dominant advective effects. Further, some important aspects related to its numerical implementation are also addressed. The ability of this methodology is illustrated, firstly, in the solution of a one-dimensional test example. Finally, the numerical simulation of a direct-chill continuous casting process is performed. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 5 Ill.
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  • 106
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 763-772 
    ISSN: 1069-8299
    Keywords: coupled vibrations ; Timoshenko beam ; boundary integral equation method ; symbolic computation ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: Symbolic computer algebra systems relieve one from the tedious task of different mathematical operations which are essential to obtain solutions. Due to their highly advanced features they have come to be used widely in computational mechanics. This paper describes an application of the modern computer algebra system Mathematica to the derivation of fundamental solutions necessary for the application of the boundary integral equation method. The problem treated is an asymmetric cross-section Timoshenko beam in free vibration. For this problem, the derivation of fundamental solutions involves lengthy mathematical operations which are very tedious if performed explicitly by hand. Therefore, using Mathematica we derive the fundamental solutions and generate the influence matrices from which the natural frequencies can be found. © 1998 John Wiley & Sons, Ltd.
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  • 107
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 751-761 
    ISSN: 1069-8299
    Keywords: acoustic ; electromagnetic ; integral equations ; scattering ; time domain ; radar cross-section ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: Analysis of high frequency scattering using pulsed illumination generates surface fields which are small over most of the scatterer most of the time. A reformulation of the usual integral equation time domain approach which exploits this is presented. It is shown that cost scaling can be reduced, with costs reduced by an order of magnitude for the examples presented, with negligible accuracy loss. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 5 Ill.
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  • 108
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 797-808 
    ISSN: 1069-8299
    Keywords: mesh equidistribution ; area preserving map ; singular BVP ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: In this paper, an adaptive mesh method is employed to solve a class of singular boundary value problems. The approach is based on an area-preserving map and some mesh shape control in two-dimensional space. Two benchmark problems, which both involve singularities in physical domains, are tested. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 6 Ill.
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  • 109
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 195-208 
    ISSN: 1069-8299
    Keywords: differential quadrature method ; elastic torsion ; numerical solution ; Poisson equation ; Laplace equation ; geometric mapping ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The governing equation of an elastic prismatic shaft is the two-dimensional Poisson equation defined on the cross-sectional area of the shaft. In this paper, the differential quadrature method (DQM) is employed to solve the Poisson equation on some non-rectangular domains. Singularities, which may appear in the expression of stress components or boundary conditions at a degenerated point of the grid, are removed by means of the Taylor expansion. The results of three examples are compared with the exact solutions. It is shown that accurate results can be achieved by the DQM. In addition, three geometric transformations are conducted in the third example so that the effect of mapping on the convergence and accuracy of results is investigated. It is found that rapid convergence can be fulfilled if the degenerated point of the mesh falls on a Dirichlet boundary. The approach addressed in the paper can be extended to other potential problems governed by either the Poisson equation or the Laplace equation. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 5 Ill.
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  • 110
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 209-218 
    ISSN: 1069-8299
    Keywords: three-dimensional convection-diffusion equation ; fourth-order compact scheme ; iterative methods ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: We present an explicit fourth-order compact finite difference scheme for approximating the three-dimensional convection-diffusion equation with variable coefficients. This 19-point formula is defined on a uniform cubic grid. We compare the advantages and implementation costs of the new scheme with the standard 7-point scheme in the context of basic iterative methods. Numerical examples are used to verify the fourth-order convergence rate of the scheme and to show that the Gauss-Seidel iterative method converges for large values of the convection coefficients. Some algebraic properties of the coefficient matrices arising from different discretization schemes are compared. We also comment on the potential use of the fourth-order compact scheme with multilevel iterative methods. © 1998 John Wiley & Sons, Ltd.
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  • 111
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 241-251 
    ISSN: 1069-8299
    Keywords: finite elements ; pollutant ; saturated porous medium ; semi-implicit method ; velocity correction ; mass transfer ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A practical problem of pollutant migration has been studied for different concentration differences and mass diffusivities using the finite element method. The results indicate that the pollutant takes years to travel 10 m into the water-saturated soil when the mass diffusivity and concentration differences are less. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 6 Ill.
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  • 112
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 277-285 
    ISSN: 1069-8299
    Keywords: numerical simulation ; steel ; quenching ; finite volume method ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: On the basis of the control volume method the algorithm and computer program for prediction of the hardness distribution in quenched steel specimens with complex geometries have been developed. The algorithm and computer program are designed to solve 2D situation problems such as the quenching of complex cylinders, cones, spheres, etc. The computer program consists of three parts: automatic computation of domain and grid generation, computation of cooling curve in grid-points, and computation of hardness in grid-points. The mathematical model has been tested experimentally. The test showed that the model describes the hardness distribution in a quenched steel specimen of a complex form, with quite satisfactory accuracy. © 1998 John Wiley & Sons, Ltd.
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  • 113
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 271-275 
    ISSN: 1069-8299
    Keywords: basis transformation ; interpolations ; finite elements ; thin plates ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The 9 df thin plate element of Mohr and Mohr uses cubic interpolation to obtain values of w at the third points of the element sides, in turn interpolating from these and the vertex values within the element. Recently this element has been modified and successfully applied to ‘potential’ problems. Subsequently it was found that the interpolations of the element of Bazeley et al. (1965, 1968) gave identical results for potential problems. In the present paper it is shown that this is because the interpolations of the two elements are exactly equivalent. © 1998 John Wiley & Sons, Ltd.
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  • 114
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 313-319 
    ISSN: 1069-8299
    Keywords: fluid mechanics ; vortex dynamics ; viscous flow ; Navier-Stokes equation ; vortex methods ; splitting procedure ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A numerical scheme has been obtained rigorously from the initial-boundary value problem for the Navier-Stokes (NS) equations in the context of the vortex method. The technique is based on a transformation of the NS equation into a parabolic equation which has an exact solution. The numerical scheme is derived by expanding the exact solution in Taylor series in powers of a small time interval. Numerical implementation is developed with use of vortex particles to represent the vortex flow domain. The method is used to solve practical engineering problems. The technique can also incorporate turbulence modelling. © 1998 John Wiley & Sons, Ltd.
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  • 115
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 347-354 
    ISSN: 1069-8299
    Keywords: thin wire loop antenna ; integro-differential equation ; frequency domain ; current distribution ; the weak formulation ; finite element technique ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The circular loop antenna is analysed by using the electric field integro-differential equation in the frequency domain. The weak form of the integro-differential is derived and then the current distribution along the circular loop antenna is calculated by solving the resulting equation via the finite element technique. Accurate results are obtained using the linear shape and test functions. © 1998 John Wiley & Sons, Ltd.
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  • 116
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 355-365 
    ISSN: 1069-8299
    Keywords: transient dynamics ; interaction of the crack sides ; crack propagation ; time domain formulation ; boundary element method ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: For two-dimensional solids the multiregion concept and the time-domain approach in the boundary element method are employed to model cracks in transient dynamics. The propagation of cracks and the closing and opening of crack sides are simulated by disconnecting and connecting degrees of freedom of a stationary mesh, as was also demonstrated recently in statics by Beer. An iterative technique is developed to determine the changes of the extent of the area where there is connection, contact or no contact at the interface between dynamically loaded regions. © 1998 John Wiley & Sons, Ltd.
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  • 117
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 941-957 
    ISSN: 1069-8299
    Keywords: geometrical non-linear ; UL formulation ; drilling degrees of freedom ; generalized conforming ; arc-length method ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: An updated Lagrangian formulation of the generalized conforming flat shell element with drilling degrees of freedom is derived based on the incremental equation of virtual work of a three-dimensional (3D) continuum for a purely geometric non-linear analysis of the space structure. While solving the non-linear equations, the Euler-Newton method and modified Euler-Newton method are used in static analyses, and the modified arc-length method and Newton arc-length method for post-buckling problems. A number of numerical examples are given to demonstrate the effectiveness of the proposed approach and programs. © 1998 John Wiley & Sons, Ltd.
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  • 118
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 473-491 
    ISSN: 1069-8299
    Keywords: temporal acceleration ; viscoelastic ; recovery ; Taylor-Galerkin ; finite elements ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper presents a finite element study based on a technique associated with time extrapolation to accelerate the convergence rate to the steady state for viscoelastic flows. The approach adopted is a local extrapolation method attributed to Neville. Temporal extrapolation is embedded within a time-marching Taylor-Galerkin/pressure-correction scheme as applied to the solution of model channel flow, 4:1 plane contraction flow and flow past a circular cylinder. In particular, consideration is given to obtaining steady-state solutions for an Oldroyd-B model. When extrapolation is performed for stress and velocity or pressure, then stress and velocity overshoot, which consequently leads to divergence. In contrast, a stable numerical scheme emerges when only the stress is extrapolated. © 1998 John Wiley & Sons, Ltd.
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  • 119
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 505-517 
    ISSN: 1069-8299
    Keywords: hypersingular integral equation ; continuous elements ; stress analysis ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: In this paper, a method for the evaluation of boundary stresses directly from the BEM solution of elastostatic problems using the static boundary integral equation is presented. The technique is valid for corners and edges as well as smooth points on the boundary, and involves defining a new interpolation function for continuous elements which incorporate certain continuity conditions arising from the hypersingular nature of the integrals involved. An integration technique based on the singularity subtraction method using series expansions is adopted for the hypersingular intergrals. Results are shown to be more accurate than those obtained with conventional techniques. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 12 Ill.
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  • 120
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    Chichester : Wiley-Blackwell
    Communications in Numerical Methods in Engineering 14 (1998), S. 539-547 
    ISSN: 1069-8299
    Keywords: boundary-only element analysis ; thermal cracking ; crack trajectory ; singular elements ; traction formula ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A boundary element procedure is developed concerning the prediction of the quasistatic crack growth in uniformly heated bimaterials. This procedure assumes the existence of an initial small crack in one of the two phases, and further cracking progress from this point due to thermal loading. The resulting mixed boundary value problem is solved by applying an incremental boundary-only method in conjunction with the multidomain technique. Fracture characterizing parameters are evaluated utilizing special crack tip singular elements and appropriate formulas. The crack path is predicted using the strain energy release rate criterion, and the mesh is updated at the end of each increment. The presented results are in good agreement with previously reported experimental results and those obtained by the finite element method. Various numerical studies were conducted and interpreted concerning crack-path dependence on individual material property mismatch. © 1998 John Wiley & Sons, Ltd.
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  • 121
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    Communications in Numerical Methods in Engineering 14 (1998), S. 559-567 
    ISSN: 1069-8299
    Keywords: gust ; buffet ; random ; vibration ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A convenient and effective finite-element-based method for buffeting analysis of complex structures is presented. This method is very efficient for structures with the natural frequencies closely spaced because their corresponding modes and the cross-correlation terms between them should be included in the mode-superposition analysis. The imperfect correlation between gust excitations can also be dealt with conveniently. A numerical example is given which shows that by using this method the buffeting analysis of complex structures with thousands of degrees of freedom, dozens of imperfectly related excitations and a number of participating modes can be executed quite easily on an ordinary IBM/486 (or 586) personal computer. © 1998 John Wiley & Sons, Ltd.
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  • 122
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    Communications in Numerical Methods in Engineering 14 (1998), S. 519-527 
    ISSN: 1069-8299
    Keywords: shell element ; large strain ; sheet metal stamping ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The paper presents a general method of large strain determination over the deformed surface of a sheet metal stamping. It is demonstrated that the conventional degenerated shell element with two normal rotation degrees of freedom is not suitable for large deformation, especially when large element rotation is present. This inaccuracy is primarily caused by the fact that the displacement field description used in the degenerated shell element is only a first-order approximation with respect to the two rotation degrees of freedom, and is therefore suitable only for small rotation angles. The new method presented in this paper replaces the two rotation DOFs with three new degrees of freedom to describe the rotation of the surface normal so that the element deformation can be accurately described with no limitation on the amount of deformation and rotation involved. The advantages of this new method are: (i) a linear and accurate expression of the displacement field in terms of nodal DOFs is obtained; (ii) the formulation is easily incorporated into any existing degenerated shell elements; (iii) the strain calculation is accurate for any amount of element rigid body rotation; (iv) if the method is used in surface grid analysis, the algorithm will not only provide correct surface strains, but also their variation through the thickness direction, i.e. the bending deformation. © 1998 John Wiley & Sons, Ltd.
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  • 123
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    Communications in Numerical Methods in Engineering 14 (1998), S. 597-608 
    ISSN: 1069-8299
    Keywords: inverse problems ; dual systems ; vibrating rod ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The discretization of the differential equation governing the axial vibration of a rod with varying cross-section leads to a specially structured matrix pencil. This paper deals with the reconstruction of this pencil from its spectrum. An iterative algorithm for this problem and an analytic characterization of complementary solutions are given. The method is demonstrated on some examples. © 1998 John Wiley & Sons, Ltd.
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  • 124
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    Communications in Numerical Methods in Engineering 14 (1998), S. 621-632 
    ISSN: 1069-8299
    Keywords: segmentation ; finite elements ; adaptive ; image ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The image segmentation problem in computer vision is considered. Given a two-dimensional domain D and a function defined on it (the original image), the problem is to obtain a ‘cartoon’ associated with this function, namely to find a set of inner boundaries which divide D into subdomains (objects) in an optimal way. The optimality criterion used here is given by the Mumford-Shah (MS) and Blake-Zisserman model, which leads to a strongly non-linear problem. Related problems appear in multiphase continuum mechanics. An iterative procedure based on an h-adaptive finite element method is proposed for the solution of this problem. The mesh adaptivity enables an efficient solution technique, with the use of basic coarse discretization and a few local regions of high resolution where needed. © 1998 John Wiley & Sons, Ltd.
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  • 125
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    Communications in Numerical Methods in Engineering 14 (1998), S. 633-645 
    ISSN: 1069-8299
    Keywords: error estimation ; boundary elements ; adaptive mesh ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A posteriori error estimation for the boundary element method is developed. The nodal error is estimated from the difference of two solutions - one from the original BEM solution, and the other from interpolation of the original solution. Based on nodal errors, a matrix analysis is carried out to calculate the corresponding errors at source points. Two formulae for estimating global percentage errors are proposed in the paper. The first formula uses nodal errors directly to estimate element error, and the second uses an integral form to calculate element error to eliminate extremely high mesh concentration near areas with singular solutions. An h-version adaptive mesh refinement is implemented to study the accuracy of the proposed error estimation. Numerical examples show that the error estimator can correctly guide mesh refinement, and a final mesh can be obtained in a few iterations. © 1998 John Wiley & Sons, Ltd.
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  • 126
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    Communications in Numerical Methods in Engineering 14 (1998), S. 647-655 
    ISSN: 1069-8299
    Keywords: finite difference methods ; stability and convergence of numerical methods ; advection schemes ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper discusses implementation strategies for second-order finite difference discretizations of advection. Purely explicit and implicit methods both have disadvantages, and we consider semi-implicit schemes in which the flux is split into a primary implicit part and a secondary explicit correction. One-dimensional scalar advection is used as a model problem to analyse the leading order error terms and the stability of the schemes. Some of the splittings turn out to be unconditionally stable, but accuracy or monotonicity may deteriorate for large time steps. © 1998 John Wiley & Sons, Ltd.
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  • 127
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    Communications in Numerical Methods in Engineering 14 (1998), S. 697-708 
    ISSN: 1069-8299
    Keywords: incompressible flow ; generalized streamline operator ; upwinding tensor ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: In the present work the backward-facing step problem is analysed in the framework of the finite element method. It is a very well-known benchmark problem for the numerical resolution of the incompressible Navier-Stokes equations. In particular, a generalized streamline operator technique (GSO) is used in the numerical approach to these equations. The results show very good agreement with those reported by other authors using different methodologies. © 1998 John Wiley & Sons, Ltd.
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  • 128
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    Communications in Numerical Methods in Engineering 14 (1998), S. 773-781 
    ISSN: 1069-8299
    Keywords: indirect address lists ; unstructured grids ; shared-memory parallel machines ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A way has been found to form indirect addressing lists in parallel on shared-memory parallel machines. The maximum possible speed-up for typical tetrahedral grids is approximately 1:23. The algorithm requires an additional scratch array to shift from the serial ‘elements surrounding points’ to the parallel ‘elements surrounding processors surrounding points’ paradigm. The algorithm developed is general in nature, i.e. applicable to all indirect addressing lists. All numerical methods requiring the construction of indirect data structures, such as sparse matrix linear algebra procedures, field and particle solvers operating on unstructured grids, and network flow applications should see a benefit from this algorithm when running on shared-memory parallel machines. © 1998 John Wiley & Sons, Ltd.
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  • 129
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    Communications in Numerical Methods in Engineering 14 (1998), S. 783-792 
    ISSN: 1069-8299
    Keywords: substructure ; frontal technique ; heat transfer ; moisture transfer ; displacement ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: This paper presents a substructuring-frontal combined algorithm for the analysis of fully coupled heat, moisture and displacement problems in unsaturated soil. The method adopted for solving the assembled matrix equations has a significant bearing on the computer storage requirement and execution time. Numerical examples are given to validate the proposed model. © 1998 John Wiley & Sons, Ltd.
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  • 130
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    Communications in Numerical Methods in Engineering 14 (1998), S. 809-819 
    ISSN: 1069-8299
    Keywords: finite element analysis ; diffusion problems ; minimum time step size ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: The solution of the equation system of the finite element method for diffusion problems is presented in the format of a sequence. The characteristics of the numerical results can thus be known through an analysis of this ‘theoretical expression’ of the solution. The characteristics of the sequence are analysed to yield expressions for the minimum time step size. © 1998 John Wiley & Sons, Ltd.
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  • 131
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    Communications in Numerical Methods in Engineering 14 (1998), S. 849-861 
    ISSN: 1069-8299
    Keywords: outgoing boundary condition ; Berkhoff ; discrete ; non-local ; surface waves ; scattering ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics , Technology
    Notes: A discrete non-local (DNL) boundary condition is used to solve the water waves propagation problem over variable depth. This condition is obtained by means of full solution of the discrete Helmholtz operator in a structured network. We consider a simulation of wave propagation around a circular island located on either a paraboloidal shoal or constant depth bathymetry. Such examples confirm the important improvement in accuracy for the DNL method over standard conditions in the near field. © 1998 John Wiley & Sons, Ltd.
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  • 132
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    Numerical Linear Algebra with Applications 5 (1998), S. 147-164 
    ISSN: 1070-5325
    Keywords: eigenvalue problem ; Lanczos method ; power method ; Krylov information ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics
    Notes: We study Lanczos and polynomial algorithms with random start for estimating an eigenvector corresponding to the largest eigenvalue of an n × n large symmetric positive definite matrix. We analyze the two error criteria: the randomized error and the randomized residual error. For the randomized error, we prove that it is not possible to get distribution-free bounds, i.e., the bounds must depend on the distribution of eigenvalues of the matrix. We supply such bounds and show that they depend on the ratio of the two largest eigenvalues. For the randomized residual error, distribution-free bounds exist and are provided in the paper. We also provide asymptotic bounds, as well as bounds depending on the ratio of the two largest eigenvalues. The bounds for the Lanczos algorithm may be helpful in a practical implementation and termination of this algorithm. © 1998 John Wiley & Sons, Ltd.
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  • 133
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    Numerical Linear Algebra with Applications 5 (1998), S. 203-217 
    ISSN: 1070-5325
    Keywords: boundary value problem ; boundary element method ; preconditioning ; fast Fourier transform ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics
    Notes: The algebraic properties of the matrix arising for the three-dimensional Dirichlet problem for Lamé equations in a rotational domain by the boundary element method are considered. The use of the special basis leads to a matrix having a block structure with sparse blocks. The possible strategies for the efficient solution of the above problem are discussed. © 1998 John Wiley & Sons, Ltd.
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  • 134
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    Numerical Linear Algebra with Applications 5 (1998), S. 299-311 
    ISSN: 1070-5325
    Keywords: iterative methods ; singular linear systems ; index of a matrix ; M-splittings ; multi-splittings ; Q-matrices ; Markov chains ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics
    Notes: Given a singular M-matrix of a linear system, convergent conditions under which iterative schemes based on M-multisplittings are studied. Two of those conditions, the index of the iteration matrix and its spectral radius are investigated and related to those of the M-matrix. Furthermore, a parallel multisplitting iteration scheme for solving singular linear systems is suggested which can be applied to practical problems such as Poisson and elasticity problems under certain boundary conditions, the Neumann problem, and in Markov chains. A discussion of that multisplitting scheme, based on Gauss-Seidel type splittings is given for computing the stationary distribution vector of Markov chains. In this case a computational viable algorithm can be constructed, since only the nonsingularity of one weighting matrix of the multisplitting is needed. © 1998 John Wiley & Sons, Ltd.
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  • 135
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    Numerical Linear Algebra with Applications 5 (1998), S. 275-297 
    ISSN: 1070-5325
    Keywords: GMRES method ; Krylov subspace methods ; non-symmetric linear systems ; homotopy curve tracking ; sparse matrix ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics
    Notes: GMRES(k) is widely used for solving non-symmetric linear systems. However, it is inadequate either when it converges only for k close to the problem size or when numerical error in the modified Gram-Schmidt process used in the GMRES orthogonalization phase dramatically affects the algorithm performance. An adaptive version of GMRES(k) which tunes the restart value k based on criteria estimating the GMRES convergence rate for the given problem is proposed here. This adaptive GMRES(k) procedure outperforms standard GMRES(k), several other GMRES-like methods, and QMR on actual large scale sparse structural mechanics postbuckling and analog circuit simulation problems. There are some applications, such as homotopy methods for high Reynolds number viscous flows, solid mechanics postbuckling analysis, and analog circuit simulation, where very high accuracy in the linear system solutions is essential. In this context, the modified Gram-Schmidt process in GMRES, can fail causing the entire GMRES iteration to fail. It is shown that the adaptive GMRES(k) with the orthogonalization performed by Householder transformations succeeds whenever GMRES(k) with the orthogonalization performed by the modified Gram-Schmidt process fails, and the extra cost of computing Householder transformations is justified for these applications. © 1998 John Wiley & Sons, Ltd.
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  • 136
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    Numerical Linear Algebra with Applications 5 (1998), S. 347-362 
    ISSN: 1070-5325
    Keywords: almost incompressible elasticity ; finite elements ; semi-coarsening refinement ; algebraic multilevel ; Engineering ; Numerical Methods and Modeling
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Mathematics
    Notes: The constant γ in the strengthened Cauchy-Buniakowski-Schwarc (CBS) inequality plays a key role in the convergence analysis of the multilevel iterative methods. We consider in this paper the approximation of the two-dimensional elasticity problem by bilinear rectangle finite elements. Two semi-coarsening refinement procedures are studied. We prove for both cases new estimates of the constant γ, uniformly on the Poisson ratio.As a result of the presented analysis we obtain an optimal order algebraic multiLevel iteration (AMLI) method for the case of balanced semi-coarsening mesh refinement. The total computational complexity of the algorithm is proportional to the size of the discrete problem with a proportionality constant independent of the Poisson ratio, that is, the algorithm is of optimal order for almost incompressible elasticity problems. Copyright © 1999 John Wiley & Sons, Ltd.
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  • 137
    ISSN: 0730-2312
    Keywords: somatostatin ; receptor isotypes ; adenylyl cyclase ; Interleukin-2 (IL-2) ; proliferation ; Jurkat cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The neuropeptide somatostatin (SRIF) modulates normal and leukemia T cell proliferation. However, neither molecular isotypes of receptors nor mechanisms involved in these somatostatin actions have been elucidated as yet. Here we show by using RT-PCR approach that mitogen-activated leukemia T cells (Jurkat) express mRNA for a single somatostatin receptor, sst3. This mRNA is apparently translated into protein since specific somatostatin binding sites (KI1 = 78 ± 3 pM) were detected in semipurified plasma membrane preparations by using 125I-Tyr1-SRIF14 as a radioligand. Moreover, somatostatin inhibits adenylyl cyclase activity with similar efficiency (IC50 = 23 ± 4 pM) thus strongly suggesting a functional coupling of sst3 receptor to this transduction pathway. The involvement of sst3 receptor in immuno-modulatory actions of somatostatin was assessed by analysis of neuropeptide effects on IL-2 secretion and on proliferation of mitogen-activated Jurkat cells. Our data show that in the concentrations comprised between 10 pM and 10 nM, somatostatin potentiates IL-2 secretion. This effect is correlated with somatostatin-dependent increase of Jurkat cell proliferation since the EC50 concentrations for both actions were almost identical (EC50 = 22 ± 9 pM and EC50 = 12 ± 1 pM for IL-2 secretion and proliferation, respectively). Altogether, these data strongly suggest that in mitogen-activated Jurkat cells, somatostatin increases cell proliferation through the increase of IL-2 secretion via a functional sst3 receptor negatively coupled to the adenylyl cyclase pathway. J. Cell. Biochem. 68:62-73, 1998. © 1998 Wiley-Liss, Inc.
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  • 138
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    Journal of Cellular Biochemistry 68 (1998), S. 74-82 
    ISSN: 0730-2312
    Keywords: cell culture ; nuclei ; nuclear degradation ; endonucleases ; polycytosine degradation ; differentiation ; cornification ; stratum corneum ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Desquamin is a glycoprotein that we have isolated from the upper granular layer and the stratum corneum of human epidermis; it is not ordinarily expressed in submerged cultures, whose terminal differentiation stops short of formation of these layers. The exogenous addition of desquamin to human cultured keratinocytes extended their maturation, and hematoxylin staining indicated a loss of cell nuclei. For confirmation, cultured cells were lysed in situ, and the nuclei were incubated with desquamin for several days, then stained with hematoxylin. Damage to the nuclei was evident: the nuclear inclusions remained intact, while the surrounding basophilic nuclear matrix was degraded. Desquamin was then tested directly for nuclease activity. Ribonuclease activity was determined by incubating desquamin with human epidermal total RNA and monitoring the dose-dependent disappearance of the 28S and 18S ribosomal RNA bands in an agarose/formaldehyde gel. On RNA-containing zymogels, we confirmed the RNase activity to be specific to desquamin. Using synthetic RNA homopolymers, we found the active RNase domains to be limited to cytosine residues. On the contrary, DNA was not degraded by an analogous procedure, even after strand-separation by denaturation. J. Cell. Biochem. 68:74-82, 1998. © 1998 Wiley-Liss, Inc.
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  • 139
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    Journal of Cellular Biochemistry 68 (1998), S. 100-109 
    ISSN: 0730-2312
    Keywords: carcinogens ; mitochondrial DNA ; nuclear DNA ; LINE ; mobile elements ; cancer ; Huntington's disease ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear DNA of normal and tumor mouse and rat tissue was examined for mitochondrial-DNA-like inserts by means of the Southern blot technique. The two probes were 32P-labeled cloned mitochondrial DNA. KpnI, which doesn't cut either mitochondrial DNA, was one of the restriction enzymes, while the enzymes that fragment mitochondrial DNA were for mouse and rat PstI and BamHI, respectively. When KpnI alone was used in the procedure a nuclear LINE family whose elements had mitochondrial-DNA-like insertions was selected. Such elements were much more abundant in tumor than in normal tissue. The results with PstI alone and BamHI alone and each combined with KpnI indicated that there were mobile LINE elements with mitochondrial-DNA-like inserts in the nuclear genome of tumor. The mouse tissues were normal liver and a transplantable lymphoid leukemic ascites cell line L1210 that had been carried for 40 years. The rat tissues were normal liver and a hepatoma freshly induced by diethylnitrosoamine in order to minimize the role of 40 years of transplantation. Our unitary hypothesis for carcinogenesis of 1971, which suggested these experiments, has been augmented to include mobile nuclear elements with inserts of mitochondrial-DNA-like sequences. Such elements have been related to diseases of genetic predisposition such as breast cancer and Huntington's disease. J. Cell. Biochem. 68:100-109, 1998. © 1998 Wiley-Liss, Inc.
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  • 140
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    Journal of Cellular Biochemistry 68 (1998), S. 121-127 
    ISSN: 0730-2312
    Keywords: heme oxygenase ; stress protein ; overexpression ; oxidative injury ; endothelial cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Heme oxygenase (HO-1) is a stress protein that has been suggested to participate in defense mechanisms against agents that induce oxidative injury such as hemoglobin/heme, hypoxia-ischemia and cytokines. Overexpression of HO-1 in endothelial cells (EC) might, therefore, protect against oxidative stress produced under these pathological conditions, by generation of CO, a vasodilator, and bilirubin, which has antioxidant properties that enhance blood vessel formation to counteract hypoxia-induced injury. A plasmid containing the cytomegalovirus promoter (pCMV) neomycin human HO-1 gene complexed to cationic liposomes, lipofectin, was used to transfect rabbit coronary microvessel EC. Cells transfected with human HO-1 gene demonstrated a twofold increase in HO activity and maintained a similar phenotype as in the nontransfected cells. Cell number in transfected cells with human HO-1 gene increased by about 45%, as compared to nontransfected or those transfected with control pCMV. Transfected and nontransfected EC revealed a similar response to basic fibroblast growth factor (bFGF) in capillary formation. However, transfected cells with the human HO-1 gene exhibited a twofold increase in blood vessel formation. The angiogenic response of EC to overexpression of HO-1 gene provides direct evidence that the inductive form of HO-1 following injury represents an important tissue adaptive mechanism for moderating the severity of cell damage produced in inflammatory reaction sites of hemorrhage, thrombosis and hypoxic-ischemia. Thus, HO-1 may participate in the regulation of EC activation, proliferation and angiogenesis. J. Cell. Biochem. 68:121-127, 1998. © 1998 Wiley-Liss, Inc.
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  • 141
    ISSN: 0730-2312
    Keywords: tyrosine phosphorylation ; insulin signaling ; tyrosine kinase ; confocal microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins. J. Cell. Biochem. 68:139-150, 1998. © 1998 Wiley-Liss, Inc.
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  • 142
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    Journal of Cellular Biochemistry 68 (1998), S. 151-163 
    ISSN: 0730-2312
    Keywords: Type I procollagen ; proto-oncogenes ; steroid ; calcitriol ; osteoblast ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Changes in the synthesis of type I collagen, the major extracellular matrix component of skin and bone, are associated with normal growth, tissue repair processes, and several pathological conditions. Expression of the COL 1A1 gene is regulated by transcriptional and post-transcriptional mechanisms. However, the hormonal regulation of type I collagen synthesis in human bone has not been well characterized. We have studied the influence of calcitriol, dexamethasone, retinoic acid, and estradiol on the COL 1A1 gene expression by determining the secretion of the C-terminal propeptide (PICP) and the levels of α1(I) procollagen mRNA in cultured human MG-63 and SaOs-2 osteoblast-like osteosarcoma cells. Similar experiments were also performed with respect to expression of the nuclear proto-oncogenes, c-fos and c-jun, in MG-63 cells.In MG-63 cells, calcitriol stimulated the synthesis and secretion of PICP. The α1(I) procollagen mRNA level was elevated with no effect on message stability, indicating a transcriptional mechanism of regulation. In contrast, dexamethasone treatment was accompanied by an accelerated rate of α1(I) procollagen mRNA turnover, observed as decreased amounts of the message and the secreted PICP, implying a posttranscriptional regulation. Retinoic acid, in turn, decreased the levels of α1(I) procollagen mRNA and secreted PICP by slowing down transcription of the COL1A1 gene without any effect on message stability. The ability of these hormones to regulate the α1(I) transcripts was sensitive to puromycin treatment, suggesting an involvement of an induced mediator protein in the action of the hormones on the COL1A1 gene. Both dexamethasone and calcitriol rapidly but transiently increased the expression of the c-fos and c-jun proto-oncogenes. Neither proto-oncogene responded to retinoic acid treatment with significant changes in mRNA levels. Estradiol treatment was found to have no influence on type I procollagen synthesis.In SaOs-2 cells, which are not as well differentiated as the MG-63 cells, calcitriol and dexamethasone did not influence type I procollagen synthesis. Retinoic acid as well as estradiol reduced collagen gene expression in these cells.These findings suggest that hormonal effects on type I procollagen synthesis may depend on the maturational state of the osteoblastic cells that express different regulatory factors and receptors, resulting in, in each case, a finely adjusted rate of gene expression. J. Cell. Biochem. 68:151-163, 1998. © 1998 Wiley-Liss, Inc.
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  • 143
    ISSN: 0730-2312
    Keywords: osteoprogenitors ; marrow-stroma ; alkaline phosphatase ; bisphosphonates ; cell proliferation ; mineralization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bisphosphonates (BPs) are inhibitors of bone resorption and soft tissue calcification. The biological effects of the BPs in calcium-related disorders are attributed mainly to their incorporation in bone, enabling direct interaction with osteoclasts and/or osteoblasts through a variety of biochemical pathways. Structural differences account for the considerable differences in the pharmacological activity of BPs. We compared the effects of two structurally different compounds, alendronate and 2-(3′-dimethylaminopyrazinio)ethylidene-1,1-bisphosphonic acid betaine (VS-6), in an osteoprogenitor differentiation system. The BPs were examined in a bone marrow stromal-cell culture system, which normally results in osteoprogenitor differentiation. The drugs were present in the cultures from days 2 to 11 of osteogenic stimulation, a period estimated as being comparable to the end of proliferation and the matrix-maturation stages. We found that the two different BPs have opposing effects on specific alkaline phosphatase (ALP) activity, on stromal-cell proliferation, and on cell-mediated mineralization. These BPs differentially interact with cell-associated phosphohydrolysis, particularly at a concentration of 10-2 of ALP Km, in which alendronate inhibits whereas VS-6 did not inhibit phosphatase activity. VS-6 treatment resulted in similar and significantly increased mineralization at 10 and 1 μM drug concentrations, respectively. In contrast, mineralization was similar to control, and significantly decreased at 10 and 1 μM drug concentrations, respectively, under alendronate treatment. J. Cell. Biochem. 68:186-194, 1998. © 1998 Wiley-Liss, Inc.
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  • 144
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    Journal of Cellular Biochemistry 68 (1998), S. 200-212 
    ISSN: 0730-2312
    Keywords: polyamines ; chromatin structure ; micrococcal nuclease ; cell cycle ; apoptosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Several studies suggest that polyamines may stabilize chromatin and play a role in its structural alterations. In line with this idea, we found here by chromatin precipitation and micrococcal nuclease (MNase) digestion analyses, that spermidine and spermine stabilize or condense the nucleosomal organization of chromatin in vitro. We then investigated the possible physiological role of polyamines in the nucleosomal organization of chromatin during the cell cycle in Chinese hamster ovary (CHO) cells deficient in ornithine decarboxylase (ODC) activity. An extended polyamine deprivation (for 4 days) was found to arrest 70% of the odc- cells in S phase. MNase digestion analyses revealed that these cells have a highly loosened and destabilized nucleosomal organization. However, no marked difference in the chromatin structure was detected between the control and polyamine-depleted cells following the synchronization of the cells at the S-phase. We also show in synchronized cells that polyamine deprivation retards the traverse of the cells through the S phase already in the first cell cycle. Depletion of polyamines had no significant effect on the nucleosomal organization of chromatin in G1-early S. The polyamine-deprived cells were also capable of condensing the nucleosomal organization of chromatin in the S/G2 phase of the cell cycle. These data indicate that polyamines do not regulate the chromatin condensation state during the cell cycle, although they might have some stabilizing effect on the chromatin structure. Polyamines may, however, play an important role in the control of S-phase progression. J. Cell Biochem. 68:200-212, 1998. © 1998 Wiley-Liss, Inc.
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  • 145
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    Journal of Cellular Biochemistry 68 (1998), S. 213-225 
    ISSN: 0730-2312
    Keywords: glutamine ; glutamate ; mitochondria ; metabolism ; HeLa cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The oxidative metabolism of glutamine in HeLa cells was investigated using intact cells and isolated mitochondria. The concentrations of the cytoplasmic amino acids were found to be aspartate, 8.0 mM; glutamate, 22.2 mM; glutamine, 11.3 mM; glycine, 9.8 mM; taurine, 2.3 mM; and alanine, 〈1 mM. Incubation of the cells with [14C]glutamine gave steady-state recoveries of 14C-label (estimated as exogenous glutamine) in the glutamine, glutamate, and aspartate pools, of 103%, 80%, and 25%, respectively, indicating that glutamine synthetase activity was absent and that a significant proportion of glutamate oxidation proceeded through aspartate aminotransferase. No label was detected in the alanine pool, suggesting that alanine aminotransferase activity was low in these cells. The clearance rate of [14C]glutamine through the cellular compartment was 65 nmol/min per mg protein. There was a 28 s delay after [14C]glutamine was added to the cell before 14C-label was incorporated into the cytoplasm, while the formation of glutamate commenced 10 s later.Aspartate was the major metabolite formed when the mitochondria were incubated in a medium containing either glutamine, glutamate, or glutamate plus malate. The transaminase inhibitor AOA inhibited both aspartate efflux from the mitochondria and respiration. The addition of 2-oxoglutarate failed to relieve glutamate plus malate respiration, indicating that 2-oxoglutarate is part of a well-coupled truncated cycle, of which aspartate aminotransferase has been shown to be a component [Parlo and Coleman (1984): J Biol Chem 259:9997-10003]. This was confirmed by the observation that, although it inhibited respiration, AOA did not affect the efflux of citrate from the mitochondria. Thus citrate does not appear to be a cycle component and is directly transported to the medium. Therefore, it was concluded that the truncated TCA cycle in HeLa cells is the result of both a low rate of citrate synthesis and an active citrate transporter. DNP (10 μM) induced a state III-like respiration only in the presence of succinate, which supports the evidence that NAD-linked dehydrogenases were not coupled to respiration, and suggests that these mitochondria may have a defect in complex I of the electron transport chain. Arising from the present results with HeLa cells and results extant in the literature, it has been proposed that a major regulating mechanism for the flux of glutamate carbon in tumour cells is the competitive inhibition exerted by 2-oxoglutarate on aspartate and alanine aminotransferases. This has been discussed and applied to the data. J. Cell. Biochem. 68:213-225, 1998. © 1998 Wiley-Liss, Inc.
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  • 146
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    Journal of Cellular Biochemistry 68 (1998), S. 247-258 
    ISSN: 0730-2312
    Keywords: SMCs ; bFGF ; collagen fibril structure ; mRNA ; atherosclerotic lesion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vascular smooth muscle cells (SMCs), the major cellular constituent of an artery, synthesize the bulk of fibrillar collagens, including type V/XI, which regulates heterotypic collagen fibril assembly. Basic fibroblast growth factor (bFGF) is a heparin-binding polypeptide growth factor that has been implicated in important events during the development of atherosclerosis, such as early intimal SMC proliferation. Here we have investigated the effects of bFGF on aortic SMC expression of type V/XI collagen. Treatment of exponentially growing or serum-deprived subconfluent cultures of bovine aortic SMCs with bFGF decreased the steady-state levels of the mRNAs for collagen type V/XI, including α1(V), α2(V), and α1(XI). The effect of bFGF was time dependent with a two- and a fourfold decrease in α2(V) mRNA observed after treatment for 24 and 48 h, respectively. This decrease resulted from a drop in the rate of α2(V) gene transcription; no change was observed in the stability of the α2(V) mRNA. Furthermore, accumulation of collagen protein decreased upon bFGF treatment. As expected, treatment with bFGF increased the rate of proliferation of serum-deprived SMCs, as judged by DNA content in the cultures, thymidine incorporation, and steady-state mRNA levels of the S-phase-expressed histone H3.2. These results suggest that bFGF plays an important role in the regulation of collagen fibril structure, with potential implications for the development and organization of an atherosclerotic lesion. J. Cell. Biochem. 68:247-258, 1998. © 1998 Wiley-Liss, Inc.
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  • 147
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    Journal of Cellular Biochemistry 68 (1998), S. 281-285 
    ISSN: 0730-2312
    Keywords: QM ; large P-antigen ; 60S ribosomal subunit ; colocalization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: QM is a human cDNA originally isolated as a transcript elevated in a nontumorigenic Wilms' tumor microcell hybrid, relative to the tumorigenic parental cell line. The QM gene encodes a 24 kDa basic protein that peripherally associates with the ribosomes. Recently, the gene for this protein has also been shown in Saccharomyces cerevisiaeto encode an essential 60S ribosomal subunit protein that is required for the joining of the 40S and 60S subunits. Since the association of QM with ribosomes can be disrupted with 1M NaCl, which has no effect on the association of core ribosomal proteins, indirect immunofluorescent cell staining was performed to colocalize the QM protein with the human large P-antigen, a core ribosomal protein of the 60S subunit, and to determine whether the assembly of the QM protein onto the 60S ribosomal subunit occurs in the nucleolus or in the cytoplasm. Our results reveal that QM co-localizes with the large P-antigen only to the cytoplasm where the rough endoplasmic reticulum is found and not to the nucleolus where ribosome assembly occurs. This finding suggests that the QM protein is most likely involved in a late step of the 60S subunit assembly and is added to the 60S ribosomal subunit in the cytoplasm and not in the nucleolus. J. Cell. Biochem. 68:281-285, 1998. © 1998 Wiley-Liss, Inc.
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  • 148
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    Journal of Cellular Biochemistry 68 (1998), S. 287-297 
    ISSN: 0730-2312
    Keywords: aorta ; mineralization ; calcification ; hydroxyapatite ; inhibitors ; arteriosclerosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mineralization of aorta is known to occur late in life and appears to be a pathological phenomenon. In vitro studies revealed that the matrix prepared from the thoracic aorta pieces after their extraction with 3% Na2HPO4 and 0.1 mM CaCl2 were mineralized under physiological conditions of temperature, pH, and ionic strength of the media to form matrix-bound mineral phase resembling hydroxyapatite in nature. However, the matrix identically prepared from the unextracted rabbits aortae failed to mineralize under identical assay conditions. The addition of the aorta extract in the assay system inhibited the above mineralization process. Standard biochemical techniques, e.g., dialysis, ion exchange, and molecular sieve chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid analysis by high-performance liquid chromatography were employed to isolate, purify, and characterize the potent inhibitory biomolecules from the aorta extract. The inhibitory activity of the aorta extract was found to be primarily due to the presence of three biomolecules having molecular weights of 66, 45, and 27-29 kDa. The above inhibitory biomolecules loosely associated with aorta may be involved in the control of calcification associated with arteriosclerosis. J. Cell. Biochem. 68:287-297, 1998. © 1998 Wiley-Liss, Inc.
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  • 149
    ISSN: 0730-2312
    Keywords: mechanical loading ; gene expression ; osteopontin ; myeloperoxidase ; rats ; differential display ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague-Dawley rats, approximately 8 months old, received either a sham or bending load using a four-point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse-transcribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4-fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4- to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function. J. Cell. Biochem. 68:355-365, 1998. © 1998 Wiley-Liss, Inc.
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  • 150
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    Journal of Cellular Biochemistry 68 (1998), S. 378-388 
    ISSN: 0730-2312
    Keywords: apoptosis ; growth suppression ; retinoic acid receptors ; ovarian cancer ; AHPN ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have used conformationally restricted retinoids to investigate the role of individual RAR subtypes and RXR in mediating the growth response of ovarian tumor cells to retinoids. Our results show that treatment of all-trans-RA-sensitive CAOV-3 cells with retinoids that bind and activate a single RAR or RXR led to a partial inhibition of growth. Treatment of all-trans-RA- resistant SKOV-3 cells did not alter growth. Maximum inhibition of growth, comparable to that observed following treatment with natural retinoids such as all-trans-RA and 9-cis-RA, was obtained only following treatment with a combination of an RAR-selective compound and an RXR-selective one. These results suggest that activation of both RAR and RXR classes is required in order to obtain maximum inhibition of ovarian tumor cell growth by retinoids. In addition, one compound, AHPN, was found to inhibit both RA-sensitive CAOV-3 and RA-resistant SKOV-3 cells. Further study of the effects of this retinoid showed that AHPN acts through an apoptotic pathway. Taken together, our results suggest that retinoids may serve as effective anti-proliferative agents in the treatment of ovarian cancer. J. Cell. Biochem. 68:378-388, 1998. © 1998 Wiley-Liss, Inc.
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  • 151
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    Journal of Cellular Biochemistry 68 (1998), S. 436-445 
    ISSN: 0730-2312
    Keywords: mouse ; PDI family proteins ; retinoic acid ; dibutyryl cAMP ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated the expression of protein disulfide isomerase family proteins (PDI, ERp61, and ERp72) in mouse F9 teratocarcinoma cells during differentiation induced by treatment with retinoic acid and dibutyryl cAMP. Each member of this family was expressed at a constitutive level in undifferentiated F9 cells. During differentiation of F9 cells to parietal or visceral endodermal cells the protein level of all these enzymes increased, although the extent of this increase in both protein and mRNA levels varied among the enzymes. Certain proteins were found to be co-immunoprecipitated with PDI, ERp61, and ERp72 in the presence of a chemical crosslinker. Type IV collagen was significantly coprecipitated with PDI whereas laminin was equally coprecipitated with the three proteins. Furthermore, 210 kDa protein characteristically coprecipitated with ERp72. Thus, the induction of PDI family proteins during the differentiation of F9 cells and their association with different proteins may implicate specific functions of each member of this family despite the common redox activity capable of catalyzing the disulfide bond formation. J. Cell. Biochem. 68:436-445, 1998. © 1998 Wiley-Liss, Inc.
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  • 152
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    Journal of Cellular Biochemistry 68 (1998), S. 427-435 
    ISSN: 0730-2312
    Keywords: α2-macroglobulin ; albumin ; placenta ; zinc ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have investigated the binding and internalization of α2-macroglobulin and serum albumin by human placental syncytiotrophoblast cells in vitro. The time course (obtained at 4°C) of α2-macroglobulin binding indicated that an equilibrium was reached after 4 h. The binding of 125I-labelled α2-macroglobulin to syncytiotrophoblast cells was competitively reduced in the presence of excess unlabelled α2-macroglobulin. When the concentration-dependence of binding was examined over a wide concentration range, non-linear regression analysis yielded a Kd of 6.4 nM. In the case of albumin, binding was weak and ligand dissociated from the cell surface during aqueous washing making it impractical to analyze the binding reaction. In other experiments, syncytiotrophoblast cells were incubated with 125I-labelled α2-macroglobulin at 37°C. Under these conditions, trypsin-resistant cell-associated radioactivity increased with time consistent with ligand internalization. 125I-Labelled-ligand was internalized with a t1/2 of about 5 min. After a lag period some radioactivity was released back into the incubation medium. When measured at times up to 210 min, this was found to consist of mostly TCA-precipitable material that had been lost from the cell surface. However, when the incubation was extended to 24 h, almost 15% of the initial cell-associated radioactivity was released to the extracellular medium as TCA-soluble material, consistent with a slow rate of ligand degradation. The specific binding of 65Zn-labelled α2M was similar to that of the 125I-labelled ligand and trypsin-resistance measurements provided evidence of α2M-mediated 65Zn uptake. These results support a role for syncytiotrophoblast in the metabolism of α2-macroglobulin during pregnancy and are also consistent with a role for α2-macroglobulin in the maternal-fetal transport of zinc. J. Cell. Biochem. 68:427-435, 1998. © 1998 Wiley-Liss, Inc.
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  • 153
    ISSN: 0730-2312
    Keywords: small heat shock proteins ; TNFα ; phosphorylation mutant ; SB203580 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The role of murine Hsp25 phosphorylation in the protection mediated by this protein against TNFα- or H2O2-mediated cytotoxicity was investigated in L929 cell lines expressing wild type (wt-) or nonphosphorylatable (mt-) Hsp25. We show that mt-Hsp25, in which the phosphorylation sites, serines 15 and 86, were replaced by alanines, is still efficient in decreasing intracellular reactive oxygen species levels and in raising glutathione cellular content, leading the protective activity of mt-Hsp25 against oxidative stress to be identical to that of wt-Hsp25. To independently investigate the role of Hsp25 phosphorylation, we blocked TNFα-induced phosphorylation of wt-Hsp25 using SB203580, a specific inhibitor of the P38 MAP kinase. This treatment did not abolish the protective activity of Hsp25 against TNFα. The pattern of Hsp25 oligomerization was also analyzed, showing mt-Hsp25 to constitutively display large native sizes, as does wt-Hsp25 after TNFα treatment in the presence of SB203580. Our results, therefore, are consistent with the possibility that the hyperaggregated form of Hsp25 is responsible for the protective activity against oxidative stress and that the phosphorylation of serines 15 and/or 86 by interfering with this structural reorganization, may lead to the inactivation of Hsp25 protective activity. J. Cell. Biochem. 69:436-452, 1998. © 1998 Wiley-Liss, Inc.
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  • 154
    ISSN: 0730-2312
    Keywords: taxol ; microtubules ; vimentin ; intermediate filaments ; protein phosphorylation ; protein kinases ; inhibitors ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Taxol, a microtubule stabilizing agent, has been extensively investigated for its antitumor activity. The cytotoxic effect of taxol is generally attributed to its antimicrotubule activity and is believed to be cell cycle dependent. Herein, we report that taxol induces hyperphosphorylation and reorganization of the vimentin intermediate filament in 9L rat brain tumor cells, in concentration- and time-dependent manner. Phosphorylation of vimentin was maximum at 10-6 M of taxol treatment for 8 h and diminished at higher (10-5 M) concentration. Enhanced phosphorylation of vimentin was detectable at 2 h treatment with 10-6 M taxol and was maximum after 12 h of treatment. Taxol-induced phosphorylation of vimentin was largely abolished in cells pretreated with staurosporine and bisindolymaleimide but was unaffected by H-89, KT-5926, SB203580, genistein, and olomoucine. Thus, protein kinase C may be involved in this process. Hyperphosphorylation of vimentin was accompanied by rounding up of cells as revealed by scanning electron microscopy. Moreover, there was a concomitant reorganization of the vimentin intermediate filament in the taxol-treated cells, whereas the microtubules and the actin microfilaments were less affected. Taken together, our data demonstrate that taxol induces hyperphosphorylation of vimentin with concomitant reorganization of the vimentin intermediate filament and that this process may be mediated via a protein kinase C signaling pathway. J. Cell Biochem. 68:472-483, 1998. © 1998 Wiley-Liss, Inc.
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  • 155
    ISSN: 0730-2312
    Keywords: transcription factor ; nuclear matrix ; YY1 ; amino acids ; functional regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The multifunctional transcription factor YY1 is associated with the nuclear matrix. In osteoblasts, the interaction of several nuclear matrix-associated transcription factors with the bone specific osteocalcin gene contributes to tissue-specific and steroid hormone-mediated transcription. A canonical nuclear matrix targeting signal (NMTS) is present in all members of the AML/CBFβ transcription factor family, but not in other transcription factors. Therefore, we defined sequences that direct YY1 (414 amino acids) to the nuclear matrix. A series of epitope tagged deletion constructs were expressed in HeLa S3 and in human Saos-2 osteosarcoma cells. Subcellular distribution was determined in whole cells and nuclear matrices in situ by immunofluorescence. We demonstrated that amino acids 257-341 in the C-terminal domain of YY1 are necessary for nuclear matrix association. We also observed that sequences within the N-terminal domain of YY1 permit weak nuclear matrix binding. Our data further suggest that the Gal4 epitope tag contains sequences that affect subcellular localization, but not targeting to the nuclear matrix. The targeted association of YY1 with the nuclear matrix provides an additional level of functional regulation for this transcription factor that can exhibit positive and negative control. J. Cell. Biochem. 68:500-510, 1998. © 1998 Wiley-Liss, Inc.
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  • 156
    ISSN: 0730-2312
    Keywords: nuclear matrix ; replication origin ; topoisomerase II-mediated DNA loop excision ; DNA loop anchorage sites ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The recently developed procedure of topoisomerase II-mediated DNA loop excision has been used to analyze the topological organization of a human genome fragment containing the gene encoding lamin B2 and the ppv1 gene. A 3.5 kb long DNA loop anchorage/topoisomerase II cleavage region was found within the area under study. This region includes the end of the lamin B2 coding unit and an intergenic region where an origin of DNA replication was previously found. These observations further corroborate the hypothesis that DNA replication origins are located at or close to DNA loop anchorage regions. J. Cell. Biochem. 69:13-18, 1998. © 1998 Wiley-Liss, Inc.
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  • 157
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    Journal of Cellular Biochemistry 69 (1998), S. 30-43 
    ISSN: 0730-2312
    Keywords: hyperthermia ; calreticulin ; chaperone complexes ; prompt glycosylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Acute heat stress leads to the glycosylation of a “prompt” stress glycoprotein, P-SG67/64, identified as calreticulin. In the present study, we used immunoprecipitation to investigate the interactions of P-SG/calreticulin with other proteins during cellular recovery from heat stress. In heat-stressed CHO and M21 cells, both glycosylated and unglycosylated P-SGs interact with HSP90, GRP94, GRP78, and the other prompt stress glycoprotein, P-SG50, in an ATP-independent manner. Specificity of HSP-P-SG interactions was determined by chemical cross-linking with the homo-bifunctional agent DSP (3,3′-dithiobis[succinimidyl propionate]). Characterization of the cross-linked complexes involving calreticulin and heat shock proteins (HSPs) showed an average mass of 400-600 kDa by gel filtration chromatography. Overall, the consistent association of glycosylated and unglycosylated calreticulin with P-SG50 and unglycosylated HSPs suggests that P-SG/calreticulin is an active member of the cast of glycone/aglycone chaperones that cooperate to achieve cellular recovery from acute heat stress. J. Cell. Biochem. 69:30-43, 1998. © 1998 Wiley-Liss, Inc.
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  • 158
    ISSN: 0730-2312
    Keywords: angiotensin II ; G proteins ; Src tyrosine kinases ; c-Fos ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Angiotensin II stimulates a biphasic activation of Raf-1, MEK, and ERK in WB liver epithelial cells. The first peak of activity is rapid and transient and is followed by a sustained phase. Angiotensin II also causes a rapid activation of p21ras in these cells. Moreover, two Src family kinases (Fyn and Yes) were activated by angiotensin II in a time- and concentration-dependent manner. Microinjection of antibodies against Fyn and Yes blocked angiotensin II-induced DNA synthesis and c-Fos expression in WB cells, indicating an obligatory involvement of these tyrosine kinases in the activation of the ERK cascade by angiotensin II. Finally, substantial reduction of the angiotensin II-stimulated activation of Fyn, Raf-1, ERK, and expression of c-Fos by pertussis toxin pretreatment argues that G proteins of the Gi family as well as the Gq family are involved in angiotensin II-mediated mitogenic pathways in WB cells. J. Cell. Biochem. 69:63-71, 1998. © 1998 Wiley-Liss, Inc.
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  • 159
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    Journal of Cellular Biochemistry 69 (1998), S. 87-93 
    ISSN: 0730-2312
    Keywords: MAP kinase pathways ; JNK ; human osteoblasts ; interleukin-1β ; UMR-106 cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We recently demonstrated the activation of extracellular signal- regulated protein kinase 1 and 2 (ERK1 and ERK2) by IGF-1, FGF-2, and PDGF-BB in normal human osteoblastic (HOB) cells as well as in rat and mouse osteoblastic cells. In this report, we have examined whether c-Jun NH2-Terminal Kinase (JNK) pathway is activated by growth factors and interleukin-1β (IL-1β) in normal HOB and rat UMR-106 cells using immune-complex kinase assay and anti-active JNK antibody, which recognizes activated forms of both JNK1 and JNK2. Results have demonstrated the presence of JNK1 and JNK2 proteins in normal HOB and UMR-106 cells. Both JNK1 and JNK2 were activated by IL-1β. IL-1β preferentially activated JNK pathway in a dose- and time-dependent manner and had little effect on ERK pathway. On the other hand, FGF-2 did not activate JNK but most strongly activated ERK pathway. The activation of JNK was maximal at 20 min whereas maximal activation of ERK1 and ERK2 was observed within 10 min. Results have clearly demonstrated that IL-1β preferentially activates JNK pathway whereas FGF-2 activates ERK pathway in normal human and rat UMR-106 osteoblastic cells. J. Cell. Biochem. 69:87-93, 1998. © 1998 Wiley-Liss, Inc.
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  • 160
    ISSN: 0730-2312
    Keywords: haemochromatosis gene ; histone gene cluster ; YACs ; cosmid contig ; sequences ; species comparison ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The HFE (HLA-H) gene is a strong candidate gene for hereditary haemochromatosis and was localized on the short arm of chromosome 6 to 6p21.3-p22. In addition, the sequence of the homologous mouse and rat cDNA and a partial sequence from the mouse gene have been reported recently. In this report, we describe the location of the human and the mouse HFE (HLA-H) gene within the histone gene clusters on the human chromosome 6 and the mouse chromosome 13. Both the human and the murine gene were located on syntenic regions within the histone gene clusters in the vicinity of the histone H1t gene. The genomic sequence of the human HFE (HLA-H) gene and the 3′ portion of the homologous mouse gene were determined. Comparison of the genomic sequences from man and mouse and the cDNA sequence from rat shows significant similarities, also beyond the transcribed region of the mouse gene. J. Cell. Biochem. 69:117-126, 1998. © 1998 Wiley-Liss, Inc.
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  • 161
    ISSN: 0730-2312
    Keywords: transcription ; mRNA stability ; dexamethasone ; gene regulation ; glucocorticoid receptor ; rat calvarial osteoblasts ; osteopontin ; vitamin D receptor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have examined the contribution of transcriptional mechanisms to the pleiotropic effects of glucocorticoids on basal and vitamin D stimulated expression of the developmentally regulated bone-specific osteocalcin (OC) gene. OC expression was systematically investigated at the level of protein, mRNA, and newly synthesized transcripts during maturation of the bone cell phenotype in cultures of fetal rat calvarial-derived osteoblasts. Our results indicate that transcriptional control of basal and hormone-regulated OC expression predominates in immature osteoblasts prior to matrix mineralization. However, in mature osteoblasts OC expression is controlled primarily by posttranscriptional mechanisms reflected by elevated mRNA levels with a decline in transcription. Vitamin D, alone or in combination with Dex, is a significant factor contributing to mRNA stabilization in mature osteoblasts with a mineralized extracellular matrix. Transcriptional modifications in response to Dex are reflected by quantitative differences between proliferating and mature osteoblasts in the formation of glucocorticoid receptor binding complexes at the proximal OC glucocorticoid response element. Vitamin D and glucocorticoid receptor mRNA levels are significantly higher in mature osteoblasts than in early stage bone cells. However, receptor complexes do not appear to be rate limiting in proliferating osteoblasts when the OC gene is not transcribed. Our results indicate (1) developmental stage-specific effects of steroid hormone on transcriptional regulation of bone expressed genes, and (2) inverse relationships between levels of transcription and cellular representation of mRNA with OC message stabilized in mature osteoblasts. J. Cell. Biochem. 69:154-168, 1998. © 1998 Wiley-Liss, Inc.
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  • 162
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    Journal of Cellular Biochemistry 69 (1998), S. 181-188 
    ISSN: 0730-2312
    Keywords: magnetic fields ; HSP70 gene expression ; human HSP70 promoter ; c-myc protein binding sites ; cellular stress ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated c-myc protein-binding sites on the HSP70 promoter as modulators of the induction of HSP70 gene expression in response to magnetic field stimulation (8μT at 60Hz) and whether the presence of c-myc protein potentiates transactivation of HSP70 expression. A 320 base pair region in the HSP70 promoter (+1 to -320) was analyzed. This region contains two c-myc-protein binding sites with consensus sequences located at -230 and -160 nucleotide positions (relative to the transcription initiation site) and overlapping with the region reported for the regulation of HSP70 gene expression by c-myc protein. This promoter region is upstream of other regulatory sequences, including the heat shock element (HSE), AP-2, and serum response element (SRE). Transfectants containing both c-myc protein-binding sites, HSP-MYC A and HSP-MYC B, and exposed to magnetic fields showed a 3.0-fold increase in expression of CAT activity as compared with sham-exposed control transfectants. Transfectants containing one c-myc binding site, HSP-MYC A, and exposed to magnetic fields showed a 2.3-fold increase in CAT expression. Transfectants in which both HSP-MYC A and HSP-MYC B binding sites were deleted showed no magnetic field sensitivity; values were virtually identical with sham-exposed controls. If the c-myc expression vector was not co-transfected with the constructs containing myc-binding sites, there was no difference in the expression of CAT activity between magnetically stimulated and sham-exposed controls, although both responded to heat shock. These data suggest that endogenous elevated levels of myc protein contribute to the induction of HSP70 in response to magnetic field stimulation. J. Cell. Biochem. 69:181-188, 1998. © 1998 Wiley-Liss, Inc.
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  • 163
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    Journal of Cellular Biochemistry 69 (1998), S. 211-220 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; protein kinase CK2 ; disulfide bonds ; sodium tetrathionate ; iodoacetamide ; sulfhydryl crosslinking ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nuclear matrix (NM) appears to be an intranuclear locale for significant and dynamic association of the ubiquitous multifunctional messenger-independent serine/threonine protein kinase CK2 that has been implicated in growth control [Tawfic et al. (1996): J Cell Biochem 61:165-171]. We have examined the nature of the association of CK2 with the NM. Nuclei prepared in the presence of a sulfhydryl-blocking reagent such as iodoacetamide demonstrate a reduction in the amount of CK2 associated with the NM to less than 5% of the control. On the other hand, when nuclei are treated with the sulfhydryl crosslinking reagent sodium tetrathionate, NM-associated CK2 increases severalfold. Treatment of nuclei with sodium tetrathionate followed by 2-mercaptoethanol blocks this increase. Nuclei isolated from rat liver and prostate behaved similarly, suggesting an identical mode of association of CK2 with the NM regardless of the organ. These results indicate a role of sulfhydryl interactions such that NM anchoring of CK2 occurs via its β subunit, which contains several vicinal cysteine residues. Further, various sulfhydryl-blocking reagents inhibited CK2 activity in a concentration-dependent manner, and the inhibitory effect was reversed by agents such as dithiothreitol, implying that cysteine residues in the CK2 play a role in its catalytic activity. J. Cell. Biochem. 69:211-220, 1998. Published 1998 Wiley-Liss, Inc.
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  • 164
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    Journal of Cellular Biochemistry 69 (1998), S. 201-210 
    ISSN: 0730-2312
    Keywords: sodium butyrate ; alkaline phosphatase ; A5 cells ; A5-DAP cells ; A5-BAG cells ; β-galactosidase ; retroviral vectors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have studied the effects of sodium butyrate (NaBu) on the expression of genes transduced by retroviral vectors and stably expressed in two salivary gland-derived cell lines, A5-DAP and A5-BAG, established earlier. These cell lines were obtained by infecting A5 cells with the retroviral vectors DAP and BAG, respectively, and by selecting neomycin-resistant transduced cells. A5-DAP cells express human placental alkaline phosphatase (PLAP) and A5-BAG cells bacterial β-galactosidase, both under the control of the viral long terminal repeat (LTR) enhancer-promoter. NaBu in the concentration of 2-8 mM inhibited the growth of A5-DAP cells, and induced the expression of heat-stable PLAP. These effects of NaBu were dose-dependent. Induction of PLAP in clones of A5-DAP cells that express different basal levels of the enzyme was not correlated with the relative inducibilty by NaBu. Exposure to 4 mM NaBu for 48 h increased the PLAP mRNA level by 31%. A5-DAP cells released, in a time-dependent manner, PLAP into the culture medium. Cells treated with NaBu released more PLAP than untreated cells in proportion to their elevated level of the enzyme. The parent A5 cells also express a low level of tissue non-specific type alkaline phosphatase, which was also induced by NaBu. NaBu inhibited the growth of A5-BAG cells also, and increased the β-galactosidase level. These data indicate the genes transduced by retroviral vectors can be induced by NaBu, which most likely interacts with the viral LTR. J. Cell. Biochem. 69:201-210, 1998. © 1998 Wiley-Liss, Inc.
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  • 165
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    Journal of Cellular Biochemistry 69 (1998), S. 221-231 
    ISSN: 0730-2312
    Keywords: sodium fluoride ; stress response ; stress proteins ; heat shock proteins ; rat brain tumor 9L cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We herein demonstrate that sodium fluoride (NaF) acts as a stress response inducer on HeLa and 9L rat brain tumor cells. NaF is only slightly cytotoxic, and inhibitory to Ser/Thr-phosphatases but not to Tyr-phosphatases in both cell lines. After treatment with 5 mM NaF for 2 h, the phosphorylation levels of vimentin and an alkali-resistant 65-kDa phosphoprotein were enhanced, a common phenomenon detected in cells under a variety of stress conditions. Under an identical treatment protocol, in which the cells were treated with 5 mM NaF for 2 h and then allowed to recover under normal growing conditions for up to 12 h, NaF differentially induced the cytoplasmic/nuclear heat-shock protein70s (including both the inducible and the constitutively expressed members of this protein family) in HeLa cells and the endoplasmic reticulum residing heat-shock protein70 (the glucose-regulated protein with an apparent molecular weight of 78 kDa) in 9L cells. Electrophoretic mobility shift assays (EMSA) using probes containing well-characterized regulatory elements revealed the activation of the heat-shock factor in HeLa but not in 9L cells; this is in good agreement with the stress protein induction pattern. Additional differential induction of binding activities toward EMSA probes individually containing NF-κB, AP-2, and CRE-like elements were detected in NaF-treated cells. The possible involvement of these binding sites as well as the corresponding factors in the stress response are discussed. J. Cell. Biochem. 69:221-231, 1998. © 1998 Wiley-Liss, Inc.
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  • 166
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    Journal of Cellular Biochemistry 70 (1998), S. 213-221 
    ISSN: 0730-2312
    Keywords: transcription ; nucleus ; cell architecture ; nuclear matrix ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: After many years of reductionistic approaches to characterize molecular mechanisms involved in transcription, the number of factors recognized to take part in this process has increased remarkably and continues to grow. When considering posttranslational modifications in conjunction with the large number of factors involved in modulating the activity of transcription complex components, the overall intricacy becomes staggering. After two decades of intensive molecular investigations, there has been a concerted effort to integrate these findings with cellular approaches to understand transcription on a more global level. This sort of reasoning actually revisits studies of approximately 20 years ago that considered the functional consequences of steroid receptor association with nuclear structure. With an abundance of new molecular probes and increasingly powerful instruments to detect them in fixed and, more recently, live cells, the issue of functional subnuclear organization is receiving increased attention. In this report, we focus on advances in characterizing the functional significance of transcription factor association with the nucleoskeleton. In particular, we consider recent biochemical and “molecular morphology” data that point to the importance of dynamic spatial and solubility partitioning of gene regulators with nuclear architecture. J. Cell. Biochem. 70:213-221, 1998. © 1998 Wiley-Liss, Inc.
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  • 167
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    Journal of Cellular Biochemistry 70 (1998), S. 222-230 
    ISSN: 0730-2312
    Keywords: functional organization of the nucleus ; nucleolus ; speckled compartment ; targeting sequence ; DNA replication ; RNA splicing ; nuclear matrix ; cell cycle ; DNA methyltransferase ; DNA ligase I ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Though there are no separating membranes within the nucleus, different factors are often concentrated at sites where their respective function is required, a phenomenum referred to as functional organization of the nucleus. How is then this organization achieved and how are the different metabolic processes integrated in the nucleus? One emerging principle was revealed by the identification of protein domains that, though not involved in catalysis, regulate enzyme activity at a higher order level by targeting enzymes to the right place at the right time. These targeting sequences constitute an assembly code for nuclear ‘protein factories,’ which ensure the extremely high efficiency and accuracy needed in a complex and competitive environment as the living mammalian cell. J. Cell. Biochem. 70:222- 230, 1998. © 1998 Wiley-Liss, Inc.
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  • 168
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    Journal of Cellular Biochemistry 70 (1998), S. 231-239 
    ISSN: 0730-2312
    Keywords: nuclear pore complexes ; nuclear localization signals ; nuclear export signals ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transport of proteins into and out of the nucleus occurs through nuclear pore complexes (NPCs) and is mediated by the interaction of transport factors with nucleoporins at the NPC. Nuclear import of proteins containing classical nuclear localization signals (NLSs) is mediated by a heterodimeric protein complex, composed of karyopherin α and β1, that docks via β1 the NLS-protein to the NPC. The GTPase Ran; the RanGDP binding protein, p10; and the RanGTP binding protein, RanBP1 are involved in translocation of the docked NLS-protein into the nucleus. Recently, new distinct nuclear import and export pathways that are mediated by members of the karyopherin β family have been discovered. Karyopherin β2 mediates import of mRNA binding proteins, whereas karyopherin β3 and β4 mediate import of a set of ribosomal proteins. Two other β karyopherin family members, CRM1 and CAS, mediate export of proteins containing leucine-rich nuclear export signals (NES) and reexport of karyopherin α, respectively. This growing family contains new members that constitute potential transport factors for cargoes yet to be identified in the future. The common features of the members of karyopherin β family are the ability to bind RanGTP and the ability to interact directly with nucleoporins at the NPC. The challenge for the future will be to identify the distinct or, perhaps, overlapping cargo(es) for each member of the karyopherin β superfamily and to characterize the molecular mechanisms of translocation of karyopherins together with their cargoes through the NPC. J. Cell. Biochem. 70:231-239, 1998.© 1998 Wiley-Liss, Inc.
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  • 169
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    Journal of Cellular Biochemistry 70 (1998), S. 240-251 
    ISSN: 0730-2312
    Keywords: actin ; actin-like proteins ; lamin ; nuclear matrix ; perinuclear actin shells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Perinuclear actin shells have been reported in a variety of organisms. The shells have been identified by staining perinuclear material with fluorescently-labelled phalloidin, but have not been localized to a specific subcellular compartment at the ultrastructural level. We show here that the shells of 3T3 cells lie in the peripheral nuclear matrix. Nuclear shells and matrix actin in other parts of the nucleus are not usually detected by immunohistochemical staining because they are inaccessible to antibodies or to phalloidin. Immunohistochemical detection of nuclear actin is only possible during its deposition at the end of mitosis, or in interphase nuclei that have been extracted with detergent, digested with nucleases and washed with high salt buffers. J. Cell. Biochem. 70:240-251, 1998. © 1998 Wiley-Liss, Inc.
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  • 170
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    Journal of Cellular Biochemistry 70 (1998), S. 281-287 
    ISSN: 0730-2312
    Keywords: retinoblastoma protein ; TATA-binding protein ; repressor ; TSA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The retinoblastoma (RB) tumour suppressor protein negatively regulates cell proliferation by modulating transcription of growth-regulatory genes. Recruitment of Rb to promoters, by association with E2F complex or by fusion with heterologous DNA-binding domains, demonstrated that Rb represses directly transcription. Recent studies also suggest that the RB protein is able to repress gene transcription mediated by the RNA polymerase I and III. Since the TATA-binding protein (TBP) is an important component for transcription mediated by all three RNA polymerases, we have analysed the functional interaction between Rb and TBP in vivo in the context of RNA pol II-driven transcription. We demonstrated that in mammalian cells Rb tethered to promoter represses TBP-mediated activation in vivo, and Rb-mediated repression is reversed in the presence of the inhibition of histone deacetylase activity by trichostatin A (TSA). J. Cell. Biochem. 70:281-287, 1998. © 1998 Wiley-Liss, Inc.
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  • 171
    ISSN: 0730-2312
    Keywords: lymphocyte ; monocyte ; cell line ; cell culture ; microgravity ; experiment development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European ‘Biorack’ provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the ‘Biorack’ facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatent in-flight), injection port, and supernatent collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatent, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground- based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities. J. Cell. Biochem. 70:252-267, 1998. © 1998 Wiley-Liss, Inc.
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  • 172
    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.
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  • 173
    ISSN: 0730-2312
    Keywords: insulin ; insulin receptor ; breast cancer cells ; insulin receptor substrate 1 ; phosphatidylinositol-3-kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In many human breast cancers and cultured cell lines, insulin receptor expression is elevated, and insulin, via its own insulin receptor, can stimulate cell growth. It has recently been demonstrated that the enzyme phosphatidylinositol-3-kinase (PI3-K) mediates various aspects of insulin receptor signaling including cell growth. In order to understand the mechanisms for insulin-stimulated cell growth in human breast cancer, we measured insulin-stimulable PI3-K activity in a non-transformed breast epithelial cell line, MCF-10A, and in two malignantly transformed cell lines, ZR-75-1 and MDA-MB157. All three cell lines express comparable amounts of insulin receptors whose tyrosine autophosphorylation is increased by insulin, and in these cell lines insulin stimulates growth. In MDA-MB157 and MCF-10A cells, insulin stimulated PI3-K activity three- to fourfold. In ZR-75-1 cells, however, insulin did not stimulate PI3-K activity. In ZR-75-1 cells PI3-K protein was present, and its activity was stimulated by epidermal growth factor, suggesting that there might be a defect in insulin receptor signaling upstream of PI3-K and downstream of the insulin receptor. Next, we studied insulin receptor substrate-1 (IRS-1), a major endogenous substrate for the insulin receptor which, when tyrosine is phosphorylated by the insulin receptor, interacts with and activates PI3-K. In ZR-75-1 cells, there were reduced levels of protein for IRS-1. In these cells, both Shc tyrosine phosphorylation and mitogen-activated protein kinase (MAP-K) activity were increased by the insulin receptor (indicating that the p21ras pathway may account for insulin-stimulated cell growth in ZR-75-1 cells).The PI3-K inhibitor LY294002 (50 μM) reduced insulin-stimulated growth in MCF-10A and MDA-MB157 cell lines, whereas it did not modify insulin effect on ZR-75-1 cell growth. The MAP-K/Erk (MEK) inhibitor PD98059 (50 μM) consistently reduced insulin-dependent growth in all three cell lines.Taken together, these data suggest that in breast cancer cells insulin may stimulate cell growth via PI3-K-dependent or-independent pathways. J. Cell. Biochem. 70:268-280, 1998. © 1998 Wiley-Liss, Inc.
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  • 174
    ISSN: 0730-2312
    Keywords: analog ; bone ; growth inhibition ; differentiation ; vitamin D ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: 1,25-Dihydroxyvitamin D3 (1,25D) is involved in the regulation of proliferation and differentiation of a variety of cell types including cancer cells. In recent years, numerous new vitamin D3 analogs have been developed in order to obtain favorable therapeutic properties. The effects of a new 20-epi analog, CB1093 (20-epi-22-ethoxy-23-yne-24a,26a,27a-trihomo-1α,25(OH)2D3), on the proliferation and differentiation of human MG-63 osteosarcoma cell line were compared here with those of the parent compound 1,25D. Proliferation of the MG-63 cells was inhibited similarly by 22%, 50% and 59% after treatment with 0.1 μM 1,25D or CB1093 for 48 h, 96 h, and 144 h, respectively. In transfection experiments, the compounds were equipotent in stimulating reporter gene activity under the control of human osteocalcin gene promoter. In cell culture experiments, however, CB1093 was more potent than 1,25D at low concentrations and more effective for a longer period of time in activating the osteocalcin gene expression at mRNA and protein levels. Also, a 6-h pretreatment and subsequent culture for up to 120 h without 1,25D or CB1093 yielded higher osteocalcin mRNA and protein levels with analog-treated cells than with 1,25D-treated cells. The electrophoretic mobility shift assay (EMSA) revealed stronger VDR-VDRE binding with analog-treated MG-63 cells than with 1,25D-treated cells. The differences in the DNA binding of 1,25D-bound vs. analog-bound VDR, however, largely disappeared when the binding reactions were performed with recombinant hVDR and hRXRβ proteins. These results demonstrate that the new analog CB1093 was equally or even more effective than 1,25D in regulating all human osteosarcoma cell functions ranging from growth inhibition to marker gene expression and that the differences in effectivity most probably resulted from interactions of the hVDR:hRXRβ-complex with additional nuclear proteins. J. Cell. Biochem. 70:414-424, 1998. © 1998 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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  • 175
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    Journal of Cellular Biochemistry 70 (1998), S. 346-353 
    ISSN: 0730-2312
    Keywords: MHC class II ; T-helper cells ; phosphotyrosine kinase ; phospholipase C-γ1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Major histocompatibility complex (MHC) class II are expressed on most activated human lymphocytes. They direct antigen presentation events in dendritic cells and B cells (collectively called antigen presenting cells), but the role for MHC class II in human T cells is not well understood. To understand the role of surface MHC class II and to identify the molecules involved in signaling, we have defined the early activation sequence in T cells when MHC class II are engaged by a specific antibody. Specifically, we have characterized the involvement of phosphotyrosine kinases, phospholipase C (PLC), and Ca2+ mobilization. With the engagement by either whole anti-class II antibody or its Fab fragments, the enzymatic activity of p56lck and ZAP-70 increased, but there was no increase in p59fyn activity. In addition, the intracellular free Ca2+ increased, which was due to enhanced influx and not to the mobilization of intracytoplasmic Ca2+. These events did not require cross-linking because they were not significantly augmented by the addition of antispecies antibody. The coimmunoprecipitation of tyrosine phosphorylated PLC-γ1 with surface MHC class II suggested that PLC-γ1 could be recruited to MHC class II after engagement. These results show the complexities of the early signals transduced by the engagement of surface MHC class II on T cells. J. Cell. Biochem. 70:346-353, 1998. © 1998 Wiley-Liss, Inc.
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  • 176
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    Journal of Cellular Biochemistry 70 (1998), S. 354-365 
    ISSN: 0730-2312
    Keywords: human fetal colon ; apolipoprotein A-I, A-IV, B-48, B-100 ; hydrocortisone ; insulin ; epidermal growth factor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The present investigation aimed at defining the localization of apolipoproteins (apo) A-I, A-IV, B-48, and B-100 along the crypt-villus axis of the human fetal colon, their biogenesis during gestation, and their hormonal regulation. Using immunofluoresence, the distribution of apo A-I and A-IV appeared as a gradient, increasing from the developing crypt to the tip of the villus. On the other hand, apo B-100 staining was found in the crypt and the lower mid-villus region with varying intensities in the upper villus cells, while the 2D8 antibody which recognizes both apo B-100 and B-48, revealed uniform staining along the crypt-villus axis. Apolipoprotein synthesis, determined by [35S] methionine labeling, immunoprecipitation, and SDS-PAGE showed a predominance of apo A-IV (53%), followed by apo A-I (23.9%), apo B-48 (13.4%), and apo B-100 (9.7%). The synthesis of each apolipoprotein was significantly modulated by hydrocortisone, insulin and epidermal growth factor (EGF). Apart from a decrease in apo B-100 exerted by EGF and a reduction in apo A-I resulting from the addition of insulin, the other apolipoproteins were all enhanced. Our data confirm that the fetal colon has the capacity to synthesize apolipoprotein A-I, A-IV, B-48, and B-100 and establish that their synthesis are modulated by hormonal and growth factors known to be involved in the regulatory mechanism of the functional development of human jejunum. J. Cell. Biochem. 70:354-365, 1998. © 1998 Wiley-Liss, Inc.
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  • 177
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    Journal of Cellular Biochemistry 70 (1998), S. 366-375 
    ISSN: 0730-2312
    Keywords: transcription ; myogenesis ; MADS domain ; DNA binding ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Previous investigations have demonstrated synergistic interactions in vivo between CCAC and A/T-rich nucleotide sequence motifs as functional components of muscle-specific transcriptional enhancers. Using CCAC and A/T-rich elements from the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding proteins that recognize these sites. Physical interactions between Sp1 and MEF-2 were demonstrated by immunological detection of both proteins in DNA binding complexes formed in vitro by nuclear extracts in the presence of only the A/T sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a glutathione-S-transferase-Sp1 fusion protein bound to glutathione beads, and by a two-hybrid assay in Saccharomyces cerevisiae. The interaction with Sp1 in vitro and in vivo is specific for MEF-2 and was not observed with serum response factor, a related MADS domain protein. Forced expression of Sp1 and MEF-2 in insect cells otherwise lacking these factors promotes synergistic transcriptional activation of a promoter containing binding sites for both proteins. These data expand the repertoire of functional and physical interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1) transcription factors that may be important for myogenic differentiation. J. Cell. Biochem. 70:366-375, 1998. © 1998 Wiley-Liss, Inc.
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  • 178
    ISSN: 0730-2312
    Keywords: anabolic ; bone ; MMP-9 ; osteoblast ; parathyroid hormone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Intermittent doses of parathyroid hormone (PTH) stimulate bone formation in animals and humans, but the molecular mechanisms underlying this phenomenon are not understood. Bone formation culminates with the expression of type I collagen, osteocalcin, and alkaline phosphatase, but genes that initiate and support the anabolic response are not known. To identify novel PTH-regulated genes in bone during the anabolic response, we used differential display-polymerase chain reaction (DDRT-PCR) to analyze RNA from young male rats injected with either human PTH (1-34) or vehicle control, once daily for 5 days. Total RNA was isolated from the distal femur metaphysis at 1, 6, and 48 h after the final injection and subjected to DDRT-PCR. We identified three PTH-responsive transcripts as matrix metalloproteinase-9 (MMP-9), creatine kinase, and the α1(I) polypeptide chain (COL1A1) of type I collagen. The concomitant upregulation of MMP-9 and COL1A1 during bone formation was particularly intriguing. Further characterization of MMP-9 expression revealed that it was localized to osteoblasts, osteocytes, megakaryocytes, and cells of the bone marrow in the rat distal femur metaphysis. Northern analysis for MMP-9 expression in other tissues indicated that this transcript was present in the kidney and brain. In vitro, PTH regulated the protein synthesis of MMP-9 by osteoblasts of the primary spongiosa. We propose that PTH may promote bone formation by mediating the subtle variation in MMP activities, thus preparing the extracellular matrix for the subsequent bone cell migration and deposition of new osteoid. J. Cell. Biochem. 70:391-401, 1998. © 1998 Wiley-Liss, Inc.
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  • 179
    ISSN: 0730-2312
    Keywords: osteopontin ; integrins ; mechano-transduction ; tyrosine kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteopontin is a predominant integrin binding protein of bone and its expression has been shown to be induced by mechanical stimuli within osteoblasts (Toma et al. [1997] J. Bone Miner. Res. 12:1626-1636). The present studies examined if the cell adhesion would mimic the mechano-transduction that stimulated opn mRNA expression and whether integrin receptors were involved in these processes. Osteopontin mRNA expression was induced three- to four-fold, 24 hours after embryonic chicken calvaria osteoblast attachment to fibronectin (FN), however no induction was observed if the cells were plated on tissue culture plastic alone. Osteopontin mRNA induction in response to cell attachment on FN was dependent on new protein synthesis and the activation of a tyrosine protein kinase(s) but unlike mechano-induction was independent of the maintenance of the cell's microfilament structure. Integrin receptor(s) were shown to be involved in mediating the signal transduction processes of both cell attachment and mechanical stimulation since incubation of osteoblasts with the integrin binding peptide RGDS partially blocked the induction of opn expression in response to both stimuli. Interestingly, incubation of the osteoblasts that were adherent on tissue culture plastic alone with the RGDS peptide lead to an induction in opn expression suggesting that integrin occupancy by itself was sufficient to initiate the signal transduction process that induced opn expression. In order to assess the role of integrin occupancy vs. focal adhesion complex formation that accompanies cell attachment, in the signal transduction process that induces opn expression, receptor clustering was stimulated pharmacologically with bombesin or lysophasphatidic acid in osteoblasts attached to tissue culture plastic. Neither compound in the absence of occupancy of the integrin receptors was capable of stimulating opn expression in attached cells, however if the cells were placed in suspension pharmacological mediation of receptor clustering and integrin occupancy were additive in their effect of inducing opn expression. These data demonstrate that induction of opn expression by mechanical stimuli and cell attachment are commonly mediated through integrin receptor(s). However, when cells are attached receptor clustering alone which accompanies focal adhesion formation was incapable of mediating signal transduction suggesting that receptor occupancy was a prerequisite to the signal transduction process that leads to the induction of opn mRNA expression. J. Cell. Biochem. 70:376-390. © 1998 Wiley-Liss, Inc.
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  • 180
    ISSN: 0730-2312
    Keywords: genome ; calmodulin ; smooth muscle ; immunohistochemistry ; heart ; development ; protein kinase ; tissue selective ; calcium ; signal transduction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We report that the genetic locus that encodes vertebrate smooth muscle and nonmuscle myosin light chain kinase (MLCK) and kinase-related protein (KRP) has a complex arrangement and a complex pattern of expression. Three proteins are encoded by 31 exons that have only one variation, that of the first exon of KRP, and the genomic locus spans approximately 100 kb of DNA. The three proteins can differ in their relative abundance and localization among tissues and with development. MLCK is a calmodulin (CaM) regulated protein kinase that phosphorylates the light chain of myosin II. The chicken has two MLCK isoforms encoded by the MLCK/KRP locus. KRP does not bind CaM and is not a protein kinase. However, KRP binds to and regulates the structure of myosin II. Thus, KRP and MLCK have the same subcellular target, the myosin II molecular motor system. We examined the tissue and cellular localization of KRP and MLCK in the chicken embryo and in adult chicken tissues. We report on the selective localization of KRP and MLCK among and within tissues and on a differential distribution of the proteins between embryonic and adult tissues. The results fill a void in our knowledge about the organization of the MLCK/KRP genetic locus, which appears to be a late evolving regulatory paradigm, and suggest an independent and complex regulation of expression of the gene products from the MLCK/KRP genetic locus that may reflect a basic principle found in other eukaryotic gene clusters that encode functionally linked proteins. J. Cell. Biochem. 70:402-413, 1998. © 1998 Wiley-Liss, Inc.
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  • 181
    ISSN: 0730-2312
    Keywords: opioids ; cathepsin D ; pS2 ; estrogen ; cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In many cancer cell lines, including breast, prostate, lung, brain, head and neck, retina, and the gastrointestinal tract, opioids decrease cell proliferation in a dose-dependent and reversible manner. Opioid and/ or other neuropeptide receptors mediate this decrease. We report that only the steroid-hormone-sensitive cell lines MCF7 and T47D respond to opioid growth inhibition in a dose-dependent manner. Therefore, an interaction of the opioid and steroid receptor system might exist, as is the case with insulin. To investigate this interaction, we have assayed two estrogen-inducible proteins (pS2 and the lysosomal enzyme cathepsin D) in MCF7 and T47D cells. When cells were grown in the presence of FBS (in which case a minimal quantity of estrogens and/ or opioids is provided by the serum), we observed either no effect of etorphine or ethylketocyclazocine (EKC) or an increase of secretion and/ or production of pS2 and cathepsin D. However, when cells were cultured in charcoal-stripped serum and in the absence of phenol red, the effect of the two opioids is different: EKC decreased the production and/ or secretion of pS2 and cathepsin D, whereas etorphine increased their synthesis and/ or secretion. The differential effect of the two general opioids was attributed to their different receptor selectivity. Furthermore, the variations of the ratio of secreted/ produced protein and the use of cycloheximide indicate that opioids selectively modify the regulatory pathway of each protein discretely. In conclusion, through the interaction with opioid and perhaps other membrane-receptor sites, opioid agonists modify in a dose-dependent manner the production and the secretion of two estrogen-regulated proteins. Opioids may therefore disturb hormonal signals mediated by the estrogen receptors. Hence, these chemicals may have potential endocrine disrupting activities. J. Cell. Biochem. 71:416-428, 1998. © 1998 Wiley-Liss, Inc.
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  • 182
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    Journal of Cellular Biochemistry 70 (1998), S. 157-158 
    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.
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  • 183
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    Journal of Cellular Biochemistry 70 (1998), S. 141-155 
    ISSN: 0730-2312
    Keywords: endothelial cell ; tyrosine phosphatase ; vanadate ; permeability ; MLCK ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The involvement of tyrosine protein phosphorylation in the regulation of endothelial cell (EC) contraction and barrier function is poorly understood. We have previously shown that myosin light chain (MLC) phosphorylation catalyzed by a novel 214 kDa EC myosin light chain kinase (MLCK) isoform is a key event in EC contraction and barrier dysfunction [Garcia et al. (1995): J Cell Physiol 163:510-522; Garcia et al. (1997): Am J Respir Cell Mol Biol 16:487-491]. In this study, we tested the hypothesis that tyrosine phosphatases participate in the regulation of EC contraction and barrier function via modulation of MLCK activity. The tyrosine phosphatase inhibitor, sodium orthovanadate (vanadate), significantly decreased electrical resistance across bovine EC monolayers and increased albumin permeability consistent with EC barrier impairment. Vanadate significantly increased EC MLC phosphorylation in a time-dependent manner (maximal increase observed at 10 min) and augmented both the MLC phosphorylation and permeability responses produced by thrombin, an agonist which rapidly increases tyrosine kinase activities. The vanadate-mediated increase in MLC phosphorylation was not associated with alterations in either phosphorylase A Ser/Thr phosphatase activities or in cytosolic [Ca2+] but was strongly associated with significant increases in EC MLCK phosphotyrosine content. These data suggest that tyrosine phosphatase activities may participate in EC contractile and barrier responses via the regulation of the tyrosine phosphorylation status of EC MLCK. J. Cell. Biochem. 70:141-155, 1998. © 1998 Wiley-Liss, Inc.
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  • 184
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    Journal of Cellular Biochemistry 70 (1998), S. 172-180 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; nuclear structure ; cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets. J. Cell. Biochem. 70:172-180, 1998. © 1998 Wiley-Liss, Inc.
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  • 185
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    Journal of Cellular Biochemistry 70 (1998), S. 193-199 
    ISSN: 0730-2312
    Keywords: parathyroid hormone-related peptide ; nucleus ; nucleolus ; intracrine actions ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: It is becoming increasingly apparent that parathyroid hormone-related peptide (PTHrP) modulates cellular function in a dual mode of action: first, by binding and activating its cognate cell surface G-protein-coupled receptor and, second, by direct intracellular effects following translocation to the nucleus and/or nucleolus of the target cell. Little is presently known about the mechanisms and events that determine the timing and degree of PTHrP nuclear translocation or the role it may serve in normal or dysregulated cellular function. Clarifying the nuclear actions of PTHrP would add significantly to our present understanding of this protein as a signaling molecule during embryonic development and as an oncoprotein whose expression in many tumors correlates with increased tumor aggressiveness and propensity for metastasis. J. Cell. Biochem. 70:193-199, 1998. © 1998 Wiley-Liss, Inc.
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  • 186
    ISSN: 0730-2312
    Keywords: gene expression ; AML/CBF transcription factors ; nuclear matrix ; cancer ; nuclear domains ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. In this article we focus on the concept that association of genes and cognate transcription factors with the nuclear matrix may support the formation and/or activities of nuclear domains that facilitate transcriptional regulation. Several lines of evidence are consistent with the concept that association of transcription factors with the nuclear matrix may be obligatory for fidelity of gene expression and maximal transcriptional activity. The identification of specific regions of transcription factors that are responsible for intranuclear trafficking to nuclear matrix-associated sites that support transcription, reinforces the linkage of nuclear structure to regulation of genes. CBFA2/AML-1 and CBFA1/AML-3 provide paradigms for directing gene regulatory factors to RNA polymerase II containing foci within the nucleus. The implications of modifications in the intranuclear trafficking of transcription factors for developmental and tissue-specific control, as well as for aberrations in gene expression that are associated with cancer and neurological disorders, are addressed. J. Cell. Biochem. 70:200-212, 1998. © 1998 Wiley-Liss, Inc.
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  • 187
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    Journal of Cellular Biochemistry 71 (1998), S. 264-276 
    ISSN: 0730-2312
    Keywords: HGF/SF ; MSH ; c-met ; tyrosinase ; B16 melanoma ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Reiterated selection in vivo of B16 murine melanoma cells for enhanced liver metastatic ability yielded a cell line (B16-LS9) dramatically overexpressing a constitutively active hepatocyte growth factor/scatter factor (HGF/SF) receptor, the product of the c-met proto-oncogene. Most likely because of their overexpressing c-met, B16-LS9 cells appear to be more responsive than parental B16-F1 cells to HGF stimulation, in terms of motility, invasion, and growth. They are also more pigmented, and express higher levels of tyrosinase as compared to parental B16-F1 cells. Therefore, we set out to explore whether HGF/SF and the liver might influence the differentiation state of B16 cells. We found that HGF/SF and MSH, two factors which reportedly have a strong influence on the phenotype and the malignant behavior of melanoma cells, may act at different levels, and with opposite results, on the regulation of gene expression. In fact, while MSH induces, at the transcriptional level, an increase in the production of both c-met and tyrosinase, HGF/SF, in contrast, promotes a decrease in the expression of both c-met and tyrosinase, however at a posttranscriptional level. These two opposite effects can counter-balance each other, when the cells are treated with both factors at the same time, apparently through a mechanism involving MAP kinase activation. The effects were, however, additive when morphological changes were considered. Most intriguingly, we also describe a very strong downregulatory activity, limited to tyrosinase expression, by hepatocytes in coculture with B16 cells. This activity, also at the posttranscriptional level, is much stronger than that exerted by HGF/SF, and appears to be due to a labile soluble factor produced by the hepatocytes. J. Cell. Biochem. 71:264-276, 1998. © 1998 Wiley-Liss, Inc.
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  • 188
    ISSN: 0730-2312
    Keywords: lysyl oxidase ; cyclooxygenase-1 ; type I collagen α1 ; prostaglandin E2 ; prostaglandin E2 receptors ; cyclic AMP ; interleukin-1β ; transforming growth factor-β ; forskolin ; 11-deoxy PGE1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In a recent communication, we demonstrated that prostaglandin E2 (PGE2) lowers basal while it ablates interleukin-1β( (IL-1β) and transforming growth factor-β (TGFβ) upregulated lysyl oxidase (LO) mRNA levels. Correspondingly, PGE2 increases cyclooxygenase-1 (COX1) mRNA in diploid, human embryo lung fibroblasts (IMR90) [Roy et al., 1996]. We now report that these actions by PGE2 are routed through cAMP via the PGE2, EP2 receptor. Among the PGE2 receptor types, the IMR90 predominantly express the EP2 mRNA. These cells also express EP3 and EP4 mRNA at comparatively low levels. Northern blot analyses show that 11-deoxy PGE1, an EP2/EP4 agonist, emulates the action of PGE2. In a similar manner to PGE2, 11-deoxy PGE1 decreases basal and TGF-β induced type I collagen α1 (COL) mRNA, basal and IL-1β induced LO mRNA while it increases COX1 mRNA. Sulprostone, an EP3/EP1 agonist, has no effect on the expression of these three genes. Forskolin, an adenylate cyclase activator, acts in a very similar manner to PGE2or 11-deoxy PGE1. It suppresses both basal and TGF-β induced COL mRNA levels. Both PGE2 and 11-deoxy PGE1 increase cAMP to a level comparable with forskolin. The role of the EP2 receptor in controlling collagen production is further underscored in the immortalized Rat-1 fibroblasts, derived from Fischer rat embryos, which do not express detectable EP2 mRNA. In these cells, PGE2 has little effect on COL mRNA level, whereas forskolin increases it. Furthermore, forskolin increases cAMP level in Rat-1 cells, whereas PGE2 does not. Overall, these results illustrate that much of the PGE2 action on the expression of COL, LO, and COX1 genes is mediated through the EP2 receptor and a subsequent increase in intracellular cAMP. J. Cell. Biochem. 71:254-263, 1998. © 1998 Wiley-Liss, Inc.
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  • 189
    ISSN: 0730-2312
    Keywords: Id ; cytokines ; hematopoiesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Hematopoietic development is regulated by a complex mixture of cytokine growth factors that guide growth and differentiation of progenitor cell populations at different stages in their development. The genetic programs that drive this process are controlled at the molecular level by the type and number of transcriptional regulators coexpressed in the cell. Both positive- and negative-acting helix-loop-helix transcription factors are expressed during hematopoietic development, with the Id-type transdominant negative regulators controlling the net helix-loop-helix activation potential in the cell at any given time. It has been demonstrated that some of these Id factors are involved in the checkpoint at which undifferentiated progenitor cells make the commitment to terminal maturation. Therefore, we sought to determine whether these Id family factors are selectively induced or extinguished by cytokines that act at different points during hematopoiesis. NFS-60, a myeloid progenitor line that proliferates in response to multiple cytokines, was stimulated by treatment with SCF, IL-3, IL-6, G-CSF, and erythropoietin. Id-1 expression correlated tightly with cellular proliferation: it declined when growth factor stimulation was withdrawn and was quickly induced whenever the cell began to proliferate. The regulation of Id-2 was more complex: its expression was slightly upregulated in factor-deprived cells but only strongly reinduced after extended exposure to cytokines that drive granulocytic differentiation (IL-6, G-CSF, and TGFβ1). These data support a cell-cycle regulatory role for Id-1 in multipotent myeloid progenitor cells and a role for Id-2 during terminal granulocytic differentiation. J. Cell. Biochem. 71:277-285, 1998. © 1998 Wiley-Liss, Inc.
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  • 190
    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: Srebrow A, Friedmann Y, Ravanpay A, Daniel CW, Bissell MJ (1998): Expression of Hoxa-1 and Hoxb-7 is regulated by extracellular matrix-dependent signals in mammary epithelial cells. J Cell Biochem 69:377-391.In Figure 3 on pages 384 and Figure 4 on page 385, two labels were misprinted. The top label on the right side of Figure 3B should have been Hoxb-7 instead of Hoxb-1, and the center label of Figure 4B should have been Hoxb-7 instead of Hoxa-7. The corrected figures are reprinted on the following pages.The Publisher apologizes for the error.
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  • 191
    ISSN: 0730-2312
    Keywords: IGFBP ; cAMP ; PKA ; prostaglandin ; bone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Insulin-like growth factor (IGF)-I and IGF-II are expressed at biologically effective levels by bone cells. Their stability and activity are modulated by coexpression of IGF binding proteins (IGFBPs). Secreted IGFBPs may partition to soluble, cell-associated, and matrix-bound compartments. Extracellular localization may sequester, store, or present IGFs to appropriate receptors. Of the six IGFBPs known, rat osteoblasts synthesize all but IGFBP-1. Of these, IGFBP-3, -4, and -5 mRNAs are induced by an increase in cAMP. Little is known about extracellular IGFBP localization in bone and nothing about IGFBP expression by nonosteoblastic periosteal bone cells. We compared basal IGFBP expression in periosteal and osteoblast bone cell cultures and assessed the effects of changes in cAMP-dependent protein kinase A or protein kinase C. Basal IGFBP gene expression differed principally in that more IGFBP-2 and -5 occurred in osteoblast cultures, and more IGFBP-3 and -6 occurred in periosteal cultures. An increase in cAMP enhanced IGFBP-3, -4, and -5 mRNA and accordingly increased soluble IGFBP-3, -4, and -5 and matrix-bound IGFBP-3 and -5 in both bone cell populations. In contrast, protein kinase C activators suppressed IGFBP-5 mRNA, and its basal protein levels remained very low. We also detected low Mr bands reactive with antisera to IGFBP-2, -3, and -5, suggesting proteolytic processing or degradation. Our studies reveal that various bone cell populations secrete and bind IGFBPs in selective ways. Importantly, inhibitory IGFBP-4 does not significantly accumulate in cell-associated compartments, even though its secretion is enhanced by cAMP. Because IGFBPs bind IGFs less tightly in cell-bound compartments, they may prolong anabolic effects by agents that increase bone cell cAMP. J. Cell. Biochem. 71:351-362, 1998. © 1998 Wiley-Liss, Inc.
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  • 192
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    Journal of Cellular Biochemistry 71 (1998), S. 382-391 
    ISSN: 0730-2312
    Keywords: dexamethasone ; bone marrow cell cultures ; IGF-I ; vertebrae ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteoblast-like cell cultures have been established from the marrow of adult rat vertebrae. We have simultaneously examined the response to dexamethasone (dex) treatment in cultures of young adult female vertebral and femoral marrow cells. Alkaline phosphatase (AP) activity was analyzed as well as the expression of mRNAs for osteocalcin (OC) and insulin-like growth factor I (IGF-I). The vertebral and femoral marrow cells were maintained for 7 days in primary culture with or without 10-8 M dex and then 6 days in secondary culture without dex or with 10-8 M or 10-7 M dex. All cells were examined on day 6 of secondary culture. Vertebral and femoral cultures each expressed the highest AP enzyme levels when grown with dex in primary culture (10-8 M) and secondary culture (10-7 M). Under all experimental conditions, vertebral cultures had lower AP enzyme activity than femoral cultures. When dex was omitted from secondary culture, OC gene expression was not detected in either vertebral or femoral passaged cells even if dex was present in primary culture. For dex conditions where OC was expressed, vertebral cultures had higher OC mRNA steady-state levels than femoral cultures. IGF-I gene expression was detected by Northern analysis in both vertebral and femoral secondary cultures. However, vertebral marrow cultures had much higher IGF-I mRNA levels compared to femoral cultures whether or not dex was present in primary culture. These findings demonstrate that dex supports the differentiation of both vertebral and femoral adult marrow osteogenic cells into osteoblasts. Our results support the hypothesis that osteoblastic marrow cultures differ depending upon which location in the skeleton they are from and that there are skeletal site-dependent differences in the insulin-like growth factor system components. J. Cell. Biochem. 71:382-391, 1998. © 1998 Wiley-Liss, Inc.
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  • 193
    ISSN: 0730-2312
    Keywords: Ishikawa cells ; endometrium ; biotin ; multinucleated cells ; predomes ; domes ; pinopods ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human Ishikawa endometrial cells form domes when confluent monolayers are stimulated with fresh fetal bovine serum. Extensive structural and biochemical changes have been detected during the approximately 30 h differentiation period. The earliest detectable change involves the formation of multinucleated structures and the appearance of “granules” that stain for biotin within those structures. Nuclei become associated with each other and are ultimately enclosed within a biotin-containing membrane. Aggregated membrane-sheathed nuclei and the cells containing them begin to elevate from the dish as biotin staining becomes apparent in apical membranes. The elevated structures are called predomes and consist of one or more very large cells containing the sheathed nuclei. Apical membranes of these unusual cells extend far out into the medium in structures that resemble endometrial pinopods. A lumen under the elevated cells fills with transcytosed fluid. As differentiation proceeds, highly concentrated chromatin material that was flattened against apical and lateral membranes of the predome cells begins to disperse. Small mononuclear cells evolve from larger predome cells. Apical membranes of predome and dome cells continue to stain for biotin. Gel electrophoresis of SDS-solubilized biotin-containing membranes, followed by Western blot analysis using avidin-linked peroxidase, resulted in three stained bands with molecular weights similar to those of the mitochondrial carboxylases: propionyl carboxylase, methylmalonyl carboxylase, and pyruvate carboxylase. J. Cell. Biochem. 71:400-415, 1998. © 1998 Wiley-Liss, Inc.
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  • 194
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    Journal of Cellular Biochemistry 72 (1998), S. 73-82 
    ISSN: 0730-2312
    Keywords: osteoblast ; bone ; stem cells ; osteoprogenitor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteoblasts are the skeletal cells responsible for synthesis, deposition, and mineralization of the extracellular matrix of bone. By mechanisms that are only beginning to be understood, stem and primitive osteoprogenitors and related mesenchymal precursors arise in the embryo and at least some appear to persist in the adult organism, where they contribute to replacement of osteoblasts in bone turnover and in fracture healing. In this paper, the nature of these cells, whether they constitute a stem cell pool or a committed progenitor pool, and aspects of their apparent plasticity are discussed. Current understanding of differential expression of osteoblast-associated genes during osteoprogenitor proliferation and differentiation to mature matrix synthesizing osteoblasts is summarized. Finally, evidence is discussed that supports the hypothesis that the mature osteoblast phenotype is heterogeneous with subpopulations of osteoblasts expressing only subsets of the known osteoblast markers, raising also the possibility of multiple parallel differentiation pathways and perhaps even different progenitor pools. J. Cell. Biochem. Suppls. 30/31:73-82, 1998. © 1998 Wiley-Liss, Inc.
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  • 195
    ISSN: 0730-2312
    Keywords: homeobox ; mammary gland ; morphogenesis ; basement membrane ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Homeobox-containing genes encode transcriptional regulators involved in cell fate and pattern formation during embryogenesis. Recently, it has become clear that their expression in continuously developing adult tissues, as well as in tumorigenesis, may be of equal importance. In the mouse mammary gland, expression patterns of several homeobox genes suggest a role in epithelial-stromal interactions. Because the stroma and the extracellular matrix (ECM) are known to influence both functional and morphological development of the mammary gland, we asked whether these genes would be expressed postnatally in the gland and also in cell lines in culture and whether they could be modulated by ECM. Using a polymerase chain reaction-base strategy five members of the Hox gene clusters a and b were shown to be expressed in cultured mouse mammary cells. Hoxa-1 and Hoxb-7 were chosen for further analysis. Hoxb-7 was chosen because it had not been described previously in the mammary gland and was modulated at different stages of gland development. Hoxa-1 was chosen because it was reported previously to be expressed only in mammary tumors, and not in normal glands. We showed that culturing the mammary epithelial cell lines SCp2 and CID-9 on a basement membrane (BM) that was previously shown to induce a lactational phenotype was necessary to turn off Hoxb-7, but a change in cell shape, brought about by culturing the cells on an inert substratum such as polyHEMA, was sufficient to downregulate Hoxa-1. This is the first report of modulation of homeobox genes by ECM. The results provide a rationale for the differential pattern of expression in vivo of Hoxa-1 and Hoxb-7 during different stages of development. The culture model should permit further in-depth analysis of the molecular mechanisms involved in how ECM signaling and homeobox genes may interact to bring about tissue organization. J. Cell. Biochem. 69:377-391, 1998. © 1998 Wiley-Liss, Inc.
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  • 196
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    Journal of Cellular Biochemistry 69 (1998), S. 1-12 
    ISSN: 0730-2312
    Keywords: two-hybrid system ; vitamin D receptor ; retinoid X receptor ; vitamin D ; protein L7 ; basic region leucine zipper domain ; coregulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The vitamin D receptor (VDR) heterodimerizes with the retinoid X receptor (RXR) and requires additional protein-protein interactions to regulate the expression of target genes. Using the yeast two-hybrid system, we identified the previously described protein L7, that specifically interacted with the VDR in the presence of vitamin D. Deletion analysis indicated, that the N-terminus of L7, which harbours a basic region leucine zipper like domain, mediated interaction with the VDR. Binding assays with purified GST-L7 demonstrated, that L7 specifically pulled down the VDR, that was either expressed in yeast or endogenously contained in the cell line U937. Interestingly, L7 inhibited ligand-dependent VDR-RXR heterodimerization, when constitutively expressed in yeast. We also demonstrate that L7 repressed binding of VDR-RXR heterodimers to a vitamin D response element. Surprisingly, L7 recruited RXR to the same response element in the presence of 9-cis retinoic acid. Ligand-dependent protein-protein interaction in the yeast two-hybrid system confirmed, that binding of L7 also was targeted at the RXR. Our data suggest, that protein L7 is a coregulator of VDR-RXR mediated transactivation of genes, that modulates transcriptional activity by interfering with binding of the receptors to genomic enhancer elements. J. Cell. Biochem. 69:1-12, 1998. © 1998 Wiley-Liss, Inc.
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  • 197
    ISSN: 0730-2312
    Keywords: genistein ; breast cancer ; p21WAF1/CIP1 ; G2/M arrest ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Genistein has been proposed to be responsible for lowering the rate of breast cancer in Asian women but the mechanism for this chemopreventive effect in vivo is unknown. In this study, we present in vitro evidence that genistein inhibits cell proliferation similarly in ER-positive and ER-negative human breast carcinoma cell lines. This inhibition is associated with specific G2/M arrest and induction of p21WAF1/CIP1 expression. Genistein results in a five- to six-fold increase in p21WAF1/CIP1 mRNA levels and a three- to four-fold increase in protein levels, only a 1.5-fold increase in p21WAF1/CIP1 transcription but a three- to six-fold increase in p21WAF1/CIP1 mRNA stability. The increase in p21WAF1/CIP1 is followed by increased apoptosis. The similar effects of genistein on a number of breast carcinoma cell lines with different ER and p53 status suggest that the actions of genistein reported here are mediated through ER and p53 independent mechanisms. The chemopreventive effects of genistein in vivo could be mediated along an identical or similar anti-proliferative pathway. J. Cell. Biochem. 69:44-54, 1998. © 1998 Wiley-Liss, Inc.
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  • 198
    ISSN: 0730-2312
    Keywords: human oocytes ; immunogold labeling ; splicing factors ; coilin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The distribution of two splicing components (snRNP and SC-35) and coilin were studied by immunogold/electron microscopy in human oocytes from antral follicles at different levels of transcriptional activity (i.e., active, intermediate, and inactive). The results showed a decrease of snRNPs and SC-35 in the karyoplasm as the oocytes progress from a transcriptionally active to the inactive state. The main areas of accumulation of both these splicing components in all stages of oocytes appeared to be the interchromatin granule clusters (IGCs). Within the IGCs, the two splicing components seemed to be spatially segregated, with the snRNPs predominantly bound to the fibrillar region, whereas the SC-35 factors are being enriched in the granular zone. The p80 coilin was found only in the nucleolus-like body (NLB), which is present in all three stages of oocytes; no coiled bodies were evident. These data are consistent with the notion that splicing occurs in the karyoplasm and that the splicing components are mobilized from a storage site (IGCs) to the site of action. J. Cell. Biochem. 69:72-80, 1998. © 1998 Wiley-Liss, Inc.
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  • 199
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    Journal of Cellular Biochemistry 68 (1998), S. 446-456 
    ISSN: 0730-2312
    Keywords: IGF-I ; IGF-II ; cAMP ; PKA ; PKC ; prostaglandin ; osteoblasts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone cells synthesize and respond to IGF-I and IGF-II which contribute to bone remodeling and linear growth. In osteoblasts, prostaglandin (PG)E2 stimulates IGF-I but not IGF-II synthesis through a cAMP-dependent protein kinase A (PKA)-related event. However, protein kinase C (PKC) activation by PGE2 enhances replication and protein synthesis by less differentiated periosteal cells more so than in osteoblast-enriched cultures from fetal rat bone. Using various PGs and other PKA and PKC pathway activators, the importance of these aspects of PGE2 activity has now been examined on IGF receptors in these bone cell culture models. PGE2 and other agents that activate PKA enhanced 125I-IGF-II binding to type 2 IGF receptors on both cell populations. In contrast, agents that activate PKC enhanced 125I-IGF-I binding to type 1 receptors on less differentiated bone cells, and of these, only phorbol myristate acetate (PMA), which activates PKC in a receptor-independent way, was effective in osteoblast-enriched cultures. No stimulator increased total type 1 receptor protein in either cell population. Consequently, ligand binding to type 1 and type 2 IGF receptors is differentially modulated by specific intracellular pathways in bone cells. Importantly, changes in apparent type 1 receptor number occur rapidly and may do so at least in part through post-translational effects. These results may help to predict new ways to manipulate autocrine or paracrine actions by IGFs in skeletal tissue. J. Cell. Biochem. 68:446-456, 1998. © 1998 Wiley-Liss, Inc.
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  • 200
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    Journal of Cellular Biochemistry 68 (1998), S. 484-499 
    ISSN: 0730-2312
    Keywords: YY1 ; zinc finger ; high-molecular-weight complex ; plasmid transfection ; nuclear matrix association ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: YY1 is a zinc finger-containing transcription factor that can both repress and activate transcription. YY1 appears to use multiple mechanisms to carry out its diverse functions. Recently, it was observed that YY1 can exist in multiple nuclear compartments. In addition to being present in the nuclear extract fraction, YY1 is also a component of the nuclear matrix. We show that YY1 can be sequestered in vivo into a high-molecular-weight complex and can be dislodged from this complex either by treatment with formamide or by incubation with an oligonucleotide containing the YY1 DNA binding site sequence. By transfecting plasmids expressing various YY1 deletion constructs and subsequent nuclear fractionation, we have identified sequences necessary for association with the nuclear matrix. These sequences (residues 256-340) co-localized with those necessary for in vivo sequestration of YY1 into the high-molecular-weight complex. We have also characterized YY1 sequences necessary for repression of activated transcription (residues 333-371) and those necessary for masking of the YY1 transactivation domain (residues 371-397). Sequences that repress activated transcription partially overlap YY1 sequences necessary for association with the nuclear matrix. However, these sequences are distinct from those that appear to mask the YY1 transactivation domain. The potential role of nuclear matrix association in controlling YY1 function is discussed. J. Cell. Biochem. 68:484-499, 1998. © 1998 Wiley-Liss, Inc.
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