Purification and characterisation of a protein kinase from winged bean

A soluble, cytoplasmic protein kinase was purified from the developing seeds of winged bean (Psophocarpus tetragonolobus) following conventional methods of protein purification including anion-exchange chromatography, gel-filtration and Blue Sepharose chromatography. The purified enzyme consists of a single polypeptide of M(r) 45,000 as determined by SDS-PAGE and gel-filtration chromatography on Sephacryl S-200. The pH optimum of the protein kinase activity was 7.0, while the optimum concentration of Mg2+ was 5 mM. The enzyme utilised casein as an exogenous phosphate acceptor. The conventional modulators of protein kinases, including the cyclic nucleotides, Ca2+ and calmodulin, did not stimulate the purified enzyme. Heparin and spermine, too, had no effect on its activity. Phosphoamino acid analysis revealed that the enzyme transferred the gamma-phosphate of ATP only to serine residues of casein. All these characteristics, taken together, classifies the purified protein kinase as a member of the casein kinase I group of enzymes.     

Iterative generation of higher-order nets in polynomial time usinglinear programming

This paper presents an algorithm for constructing and training a class of higher-order perceptrons for classification problems. The method uses linear programming models to construct and train the net. Its polynomial time complexity is proven and computational results are provided for several well-known problems. In all cases, very small nets were created compared to those reported in other computational studies.     

New galvanic cell designs for minimizing electrode polarization

New galvanic cell designs, incorporating one or two buffer electrodes, are developed to minimize the electrode polarization caused by electrochemical permeability of the electrolyte at high temperature. When a nonpolarizable reference electrode is employed, a cell with three-electrode compartments can be used to measure the chemical potential of oxygen in two-phase fields of ternary systems, associated with one degree of freedom at constant temperature. A buffer electrode is placed between the reference and measuring electrodes. The buffer electrode, maintained at approximately the same oxygen chemical potential as the measuring electrode, absorbs the semipermeability flux of oxygen between reference and measuring electrodes. When the reference electrode is polarizable, two buffer electrodes are required between the reference and measuring electrodes. The reference and reference-buffer electrodes have the same chemical potential of the active species. Similarly the measuring electrode and its buffer are of approximately the same chemical potential. A significant chemical potential difference exists only between the two buffers, which may become polarized due to coupled transport of ions and electronic defects through the electrolyte. Since the reference and measuring electrodes are insulated, the emf of the solid state cell is unaffected. The use of the buffer electrode designs permit more accurate thermodynamic measurements on metal and ceramic systems at high temperature.     

Some aspects of the precipitation of metastable intermetallic phases in INCONEL 718

Some aspects of the precipitation of the metastable intermetallic phases —γ″ and γ″—in the commercial nickel base superalloy, INCONEL 718, have been investigated over a wide range of aging temperatures. It has been confirmed that the spherical γ″ particles and the ellipsoidal γ″ particles evolve predominantly through homogeneous nucleation. Precipitation of the former does not appear to precede that of the latter in this alloy. The tetragonal distortion associated with the γ″ particles has been found to increase with increasing precipitate size. It has been observed that at certain temperatures, physical association between precipitates of the two types occurs frequently, leading to the development of different composite precipitate morphologies. During coarsening, the precipitate size has been found to depend linearly on the cube root of the aging time for γ′ as well as γ″ particles.     

Gradient solid electrolytes for thermodynamic measurements: System Na2CO3-Na2SO4

The thermodynamic properties of Na₂CO₃-Na₂SO₄ solid solution with hexagonal structure have been measured in the temperature range of 873 to 1073 K, using a composite-gradient solid electrolyte. The cell used can be represented as Pt, CO₂′ + O₂∥ Na₂CO₃Na₂ (CO₃)_x(SO₄)_1-x) ∥ CO₂″ + O₂″, Pt The composite-gradient solid electrolyte consisted of pure Na²CO³ at one extremity and the solid solution under study at the other, with variation in composition across the electrolyte. A CO₂ + O₂ + Ar gas mixture was used to fix the chemical potential of sodium at each electrode. The Nernstian response of the cell to changes in partial pressures of CO₂ and O₂ at the electrodes has been demonstrated. The activity of Na₂CO₃ in the solid solution was measured by two techniques. In the first method, the electromotive force (emf) of the cell was measured with the same CO₂ + O₂ + Ar mixture at both electrodes. The resultant emf is directly related to the activity of Na₂CO₃ at the solid solution electrode. By the second approach, the activity was calculated from the difference in compositions of CO₂ + O₂ + Ar mixtures at the two electrodes required to produce a null emf. Both methods gave identical results. The second method is more suitable for gradient solid electrolytes that exhibit significant electronic conduction. The activity of Na₂CO₃ exhibits positive deviation from Raoult’s law. The excess Gibbs’ energy of mixing of the solid solution can be represented using a subregular solution model such as the following: ΔG^E = X(1 - X)[6500(±200)X + 3320(±80)(l - X)] J mol^-1 whereX is the mole fraction of Na₂CO₃. By combining this information with the phase diagram, mixing properties of the liquid phase are obtained.     

Vascular permeability factor/vascular endothelial growth factor-mediated signaling in mouse mesentery vascular endothelium

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a multifunctional cytokine and growth factor that has important roles in both pathological and physiological angiogenesis. VPF/VEGF induces vascular hyperpermeability, cell division, and other activities by interacting with two specific receptor tyrosine kinases, KDR/Flk-1 and Flt-1, that are selectively expressed on vascular endothelium. The signaling cascade that follows VPF/VEGF interaction with cultured endothelium is only partially understood but is known to result in increased intracellular calcium, activation of protein kinase C, and tyrosine phosphorylations of both receptors, phospholipase C-gamma (PLC-gamma) and phosphatidylinositol 3'-kinase. For many reasons, signaling events elicited in cultured endothelium may not mimic mediator effects on intact normal or tumor-induced microvessels in vivo. Therefore, we developed a system that would allow measurement of VPF/VEGF-induced signaling on intact microvessels. We used mouse mesentery, a tissue whose numerous microvessels are highly responsive to VPF/VEGF and that we found to express Flk-1 and Flt-1 selectively. At intervals after injecting VPF/VEGF i.p., mesenteries were harvested, extracted, and immunoprecipitated. Immunoblots confirmed that VPF/VEGF induced tyrosine phosphorylation of several proteins in mesenteric microvessels as in cultured endothelium: Flk-1; PLC-gamma; and mitogen-activated protein kinase. Similar phosphorylations were observed when mesentery was exposed to VPF/VEGF in vitro, or when mesenteries were harvested from mice bearing the mouse ovarian tumor ascites tumor, which itself secretes abundant VPF/VEGF. Other experiments further elucidated the VPF/VEGF signaling pathway, demonstrating phosphorylation of both PYK2 and focal adhesion kinase, activation of c-jun-NH2-kinase with phosphorylation of c-Jun, and an association between Flk-1 and PLC-gamma. In addition, we demonstrated translocation of mitogen-activated protein kinase to the cell nucleus in cultured endothelium. Taken together, these experiments describe a new model system with the potential for investigating signaling events in response to diverse mediators on intact microvessels in vivo and have further elucidated the VPF/VEGF signaling cascade.     

Evolution and thermal stability of Ni3V and Ni2V phases in a Ni-29 at. pct V alloy

Eutectoid decomposition of the disordered fcc Ni-V solid solution in the composition range of 25 to 33.3 at. pct V gives rise to a mixture of the ordered Ni₃V and Ni₂V phases. In the present work, the evolution and thermal stability of these phases were studied in a Ni-29 at. pct V alloy. Solution-treated and water-quenched specimens, when aged at 850 °C, were found to exhibit two types of microstructure. In the first, the Ni₂V phase precipitated in a lamellar Ni₃V matrix where a pair of conjugate lamellae corresponded to two variants of the Ni₃V phase. In the second morphology, the Ni₂V phase precipitated within a Ni₃V matrix comprising a single variant of the Ni₃V phase. The Ni₂V phase was observed to precipitate in a plate-shaped morphology, exhibiting {120}_fcc-type, habit planes. The precipitation of the Ni₂V plates in the Ni₃V lamellae resulted in zigzag interfaces between adjacent Ni₃V domains. Both the microstructures were found to be thermally quite stable and did not coarsen appreciably on prolonged aging. However, the prolonged aging caused the renucleation of the Ni₃V and the Ni₂V phases in the vicinity of the grain boundaries in a manner similar to “recrystallization.” The stability of the aged microstructure could be attributed to the nature of the interfaces between different domains of the Ni₃V and Ni₂V phases.     

Structure and strength of dilute zirconium-copper martensites

The morphology, the substructure and the strength of dilute zirconium-copper martensites were examined. The martensites were of the dislocated lath type. The precipitation of an intermetallic phase at the lath boundaries could not be suppressed even by rapid beta-quenching. The strength of these martensites was substantially enhanced with increasing copper additions. This increased strength appeared to be due both to solid solution hardening and the distribution of precipitates at the lath boundaries. The work-hardening behaviour of these martensites could be analysed in terms of Ashby's theory of the deformation of plastically inhomogeneous materials.     

Study of the effect of static/dynamic Coulomb friction variation at the tape-head interface of a spacecraft tape recorder by non-linear time response simulation

NASA's Voyager 1977 mission to Jupiter-Saturn utilizes a magnetic digital tape recorder (DTR) on each of the two flyby spacecrafts. It has been experimentally determined that about 97% of the total output torque of the drive motor of the Voyager DTR's is required to overcome the static/dynamic Coulomb friction at the tape-head interface while the remaining 3% of the torque is utilized to overcome other bearing and aerodynamic frictions. Needless to say, the accuracy of the analytical simulation of the DTR servo depends to a very great extent on the fidelity with which the analytical model of the highly non-linear and piece-wise continuous static-dynamic Coulomb friction represents real life. In order to demonstrate the power and flexibility of graphic interactive simulation in time domain, three aspects of a realistic Coulomb friction model (viz., the static component, the dynamic component and the general shape) were interactively adjusted with a view to determining the sensitivity of the system behavior to such perturbations. A number of stable system configurations were arrived at, which had significantly different static/dynamlc components of Coulomb friction than current (nominal) design values.