Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate-water oxygen exchange, using (18)O-substituted GTP and inorganic phosphate (P(i)), was used to determine the extent of reversal of the hydrolysis step and of P(i) binding. The data show that neurofibromin and P(i) dissociate from the NF1.Ras.GDP.P(i) complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P(i) release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P(i) release causes the large change in affinity of neurofibromin, which then dissociates rapidly.

Novel guanine nucleotide analogues have been used to investigate the role of Mg(2+) in nucleotide release and binding with the small G protein rac. The fluorescent analogues have 7-(ethylamino)-8-bromocoumarin-3-carboxylic acid attached to the 3'-position of the ribose via an ethylenediamine linker. This modification has only small effects on the interaction with rac. There are large fluorescence changes on binding of the triphosphate to rac, on hydrolysis, and then on release of the diphosphate. Furthermore, the fluorescence is sensitive to the presence of Mg(2+) in the active site. Using this signal, it was shown that, for a variety of conditions, the nucleotides dissociate by a two-step mechanism. Mg(2+) is released first followed by the nucleotide. With the diphosphate, Mg(2+) is fast and nucleotide release slow. For the fluorescent GMPPNP analogue, the rate of dissociation is limited by Mg(2+) release. In the latter case, Mg(2+) binds tightly with a K(d) of 61 nM, whereas for the diphosphate the K(d) is 11 microM (30 degrees C, pH 7.6).

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Looking to string education as a model, suggestions for reorganisation of learning experiences, academic structure and assessment are discussed. Medical educators are encouraged to think about the challenges they face in creative ways. By looking outside traditional medical education, solutions may be found to new and old educational dilemmas.

A comparison of the effectiveness of two educational activities are carried out; a standard on-curricular postgraduate unit and an off-curricular school activity. The taught area for both is the theme of enterprise and entrepreneurship. They share similar intended learning outcomes, equipping participants with the same skills, knowledge and tools to set up their own business start-up. Another similarity is the number of contact hours with the students, however the main difference is the span these two activities take place; over a full semester or over a week. A survey was designed to be used in post teaching sessions to evaluate the effectiveness and impact the activities had on the students in the area of enterprise and entrepreneurship. The results indicated that both activities had an impact on start-up intention and activity however, the cost of the enterprise school per head and the fact that the on-curricular activities provide a recognized qualification in case the business fails means that short intensive off-curricular activities are better suited for researchers with limited time and who are still open minded about career choices. Both activities serve their own purpose and value as they provide the desired interventions for supporting positive attitudes towards enterprise and entrepreneurship.