Photoaffinity labeling and photoaffinity cross-linking of phosphofructokinase-1 from Saccharomyces cerevisiae by 8-azidoadeninenucleotides.

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alpha- and four beta-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, such as 8-azidoadenosine 5'-triphosphate, 8-azido-1,N6-ethenoadenosine 5'-triphosphate, and 8-N3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation. All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis revealed distinctly differences between them. Photolabeling causes a decrease in enzyme activity to a similar extent, and ATP acts as competitive effector to inactivation. Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A, monoepsilon-8-diN3AP4A, and diepsilon-8-diN3AP4A) were applied for studying the spatial arrangement of the nucleotide binding sites. No cross-linking of the subunits was obtained by irradiation of the enzyme with 8-diN3AP4A. Photolabeling with diepsilon-8-diN3AP4A resulted in the formation of two alpha-beta cross-links with different mobilities in the SDS-polyacrylamide gel electrophoresis, while monoepsilon-8-diN3AP4A yielded only one alpha-beta cross-link. Because an interfacial location of the catalytic sites between two subunits is less likely, we suggest that the formation of cross-linked subunits may be the result of specific interactions of the bifunctional photolabels with regulatory sites at the interface of both subunits.

Cooperative image workstation based on explicit models of diagnostic information requirements.

A medical image workstation designed to act as a cooperative dialogue partner in diagnostic radiology has been conceived, and a prototype has been made. The system can automatically select relevant information (eg, from current and previous examinations) and generate a meaningful and appropriate image arrangement on the display screen. For a number of routinely performed tasks in radiology, the users' interaction may be as simple as switching from one patient to the next. This is shown to considerably simplify and speed up radiological image access and presentation, saving the user time and effort. The cooperative system response is based on explicit (formalized and computer-accessible) models of diagnostic information requirements. These models are context dependent and take into account that diagnostic information needs vary with radiological work procedures, workstation users, and patient cases. Initial models have been acquired from expert radiologists in two European hospitals and were integrated in a cooperative workstation prototype. For the representation of models, rule-based and object-oriented techniques were applied. The rule base was designed with a distinct modular structure, separating between rule sets for general, task-dependent, and user-dependent information requirements. The installed rule-based mechanism also offers a solution for the automatic prefetching of images to avoid transmission delays in the course of diagnostic work sessions. The first part of the report reviews the objectives for the design of cooperative workstation user interfaces and explains the benefits from the users' point of view. In the second part, the acquisition, structuring, formalization, and representation of context-dependent information requirement models is described. The rule-based model is explained using examples. A layered workstation architecture consisting of model, object, and real-time layers is presented. Difficulties in the implementation of cooperative workstations are discussed that point to future research topics and standardization efforts.