Showing papers by "David E. Shaw published in 1999"

Method and apparatus for delivering electronic advocacy messages

Abstract: A method of operating an advocacy network (2000) is provided One or more users (2110, 2120) are selected based on information in a user database (2330) containing, for example, demographic or psychographic information about users (2110, 2120) as well as information identifying a representative (2400) associated with each user (2110, 2120) such as a home address An advocacy message (2500) is sent to a user through a first communication network (2200), such as by sending an advocacy message associated with an e-mail message The advocacy message (2500) may include a portion that lets the user generate an indication, such as by clicking on a portion of a computer screen When an indication is received, a response message (2600) is sent to the representative (2400) associated with the user (2110, 2120) The response message (2600) includes information about the user (2110, 2120), such as the user's name and home address, and may be sent at a pre-determined time along with response messages (2600) from other users (2110, 2120)

Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254.

Abstract: Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates (Kitchens et al., 1999). We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance. In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2′-deoxyuridine (FdUrd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K m for dUMP and k cat were not substantially different among the variants and were comparable with the wild-type values, but K m for methylenetetrahydrofolate (CH 2 H 4 PteGlu) was >10-fold higher for P254D. Values of k on and k off for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants. Drastic differences were observed for CH 2 H 4 PteGlu binding, with K d values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH 2 H 4 PteGlu correlates well with K i values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation. The crystal structure of P254S was determined at 1.5 A resolution and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40°, and the side chain forms an ion pair with Arg-225.