Implementing cognitive learning strategies in computer-based educational technology: a proposed system.

Switching the development focus of computer-based instruction from the concerns of delivery technology to the fundamentals of instructional methodology, is a notion that has received increased attention among educational theorists and instructional designers over the last several years. Building upon this precept, a proposed methodology and computer support system is presented for distilling educational objectives into concept maps using strategies derived from cognitive theory. Our system design allows for a flexible and extensible architecture in which an educator can create instructional modules that encapsulate their teaching strategies, and mimics the adaptive behavior used by experienced instructors in teaching complex educational objectives.

Use of an oriented transmembrane protein to probe the assembly of a supported phospholipid bilayer.

Planar-supported phospholipid bilayers formed by the adsorption of vesicles are increasingly used in the investigation of lipid-dependent reactions. We have studied the way in which these bilayers are formed with phospholipid vesicles containing the transmembrane protein Tissue Factor (TF). TF complexed with the serine protease, factor VIIa, is the primary initiator of blood coagulation by way of activation of the zymogen factor X. TF has been shown to orient randomly on the inner and outer leaflets of vesicles. We used proteolytic digestion to produce vesicles in which the extracellular domain of TF is located on the inner leaflet. These vesicles show no cofactor activity for factor VIIa as a result of the inability of the extracellular domain of TF to bind VIIa. After freeze/thawing, 50% of the cofactor activity was regained, indicating reorientation of the sequestered, inner leaflet TF. Adsorption of these vesicles to the inner surface of glass microcapillaries results in a continuous phospholipid bilayer. The microcapillaries were perfused with a solution of factors VIIa and X, and the effluent was monitored for factor Xa production, a sensitive measure of the activity of the TF-VIIa complex. For coatings produced with the digested vesicles, minimal TF-VIIa activity was observed, showing that the supported bilayer preserves the orientation of the leaflets in the vesicles, i.e., the outer leaflet of the vesicles forms the outer leaflet of the supported bilayer.

Transport rate limited catalysis on macroscopic surfaces: the activation of factor X in a continuous flow enzyme reactor.

Blood coagulation is initiated on cells which present a macroscopic surface to the flowing blood stream. We have used a continuous flow enzyme reactor to model this system and to investigate the effects of shear rate and mass transport on the activation of factor X by the complex of the transmembrane protein, tissue factor, and the serine protease, factor VIIa. This initial step of blood coagulation was found to be half-maximal at very low enzyme densities (0.03-0.06%) on the wall of the capillaries. In agreement with hydrodynamic theory, the apparent Km in the flow reactor was correlated with the cube root of the wall shear rate. These data indicate that at high tissue factor densities (> 0.6%) the activation of 150 nM factor X is controlled by the flux of X toward the surface, which is controlled by wall shear rate and substrate concentration. The appearance of the product, Xa, in the effluent was delayed to 8-12 min, which was caused by high-affinity binding of Xa to the phospholipid. This delay was considerably shortened by embedding tissue factor into PC or by coating the PS/PC surface with the phospholipid binding protein, annexin V. At low tissue factor densities, annexin V inhibited X activation by 45%, while no inhibition was observed at high densities. We demonstrate that when the reaction is limited by substrate flux, addition of further enzyme does not increase reaction rates. This contrasts with classical three-dimensional catalysis in which the initial velocity is ordinarily linear with the enzyme concentration.

Flow dependence of factor X activation by tissue factor-factor VIIa.

Blood coagulation is initiated after blood is exposed to the transmembrane glycoprotein Tissue Factor (TF). The continuous flow reactor has been used to study the flow-dependent activation of factor X by the complex of TF and factor VIIa. The apparent Km value of X for TF-VIIa in vesicles of 16 nM is low compared to the value of > 600 nM on a macroscopic surface. At 17 to 70 fmoles TF-VIIa/cm2, the steady state Xa production rate was fairly insensitive to the enzyme density, which is in contrast with classical Michaelis-Menten kinetics. The X activation rate was linear with the cube root of the flow rate. These results show that X activation is limited by transport of factor X across a boundary layer towards the enzyme complex.

Rotamer-specific fluorescence quenching in tyrosinamide: Dynamic and static interactions.

We have examined the environments of the three phenol rotamers about the C(α)-C(ß) bond in tyrosinamide by fluorescence quenching. Steady-state acrylamide quenching yields a nonlinear stern-Volmer plot. With three distinct emitting species and no other information about the system, it is impossible to analyze the data due to the number of variables which have to be determined. We therefore reduced the number of variables by independently determining the fractional intensity and dynamic quenching constant for each rotamer through time-resolved fluorescence quenching studies. These parameters were then used to analyze the steady-state data for any contribution of static quenching. We conclude that the nonlinear Stern-Volmer plot for the quenching of tyrosinamide by acrylamide is a consequence of each rotamer having a distinct dynamic quenching constant and the presence of static quenching. The static quenching can be represented by either the sphere-of-action model involving two of the three rotamers or the ground-state complex model involving all three rotamers.