Screening and characterization of estrogenic activity from a hydroxystilbene library.

BACKGROUND: Compounds that either inhibit or induce an estrogen response in vivo are important as potential drugs and biochemical tools. Non-steroidal stilbene analogs such as tamoxifen are known to function as both estrogen agonists and antagonists depending upon the analog structure. This family of compounds is amenable to parallel-manifold synthesis because stilbene analogs are easily synthesized using a single-step olefination reaction. RESULTS: We have prepared a small 23-component hydroxystilbene library using a solid phase synthesis approach. The library was screened for estrogenic and antiestrogenic activity using a cell-based bioassay that measures estrogen receptor-mediated transcription of a reporter gene. Three of the analogs proved to have dose-dependent estrogenic activity with EC50 values between 5 microM and 15 microM. Further characterization of the hydroxystilbene-mediated estrogenic activity suggests that the agonist activity results from direct binding to the steroid site on the estrogen receptor with IC50 values of 1-10 microM. CONCLUSIONS: The results of this study show that classic olefination chemistry can be adapted to a solid-phase format for parallel synthesis of analog libraries. Although yields varied for the individual analogs, sufficient quantity of pure material was obtained directly from the resin for structural characterization and biological evaluation. This study further validates solid-phase organic synthesis as a useful approach for rapid parallel-manifold library synthesis to augment both lead compound discovery and optimization.

Paradoxical production of target protein using antisense RNA expression vectors.

We used antisense RNA in a protocol designed to reduce estrogen receptor (ER) content in human breast cancer cells and observed paradoxical increases in ER levels. ER protein activity was measured using a highly sensitive reporter gene assay that relies on the ability of functional ER to bind a consensus estrogen response element (ERE) and drive the production of chloramphenicol acetyl-transferase (CAT). Upon transient transfection of ER-positive cell lines with three different vectors containing the full-length ER cDNA cloned in an antisense orientation, we observed unexpected increases in ER-driven CAT activity. To further investigate this phenomenon, expression from the antisense ER vectors was studied in an ER-negative breast tumor cell line, MDA-MB-453. ER activity was observed in these ER-negative cells upon transient transfection with each of three antisense ER vectors, but not from control vectors. Expression of ER from antisense constructs was 30-100-times less efficient than ER expression from isogenic sense constructs. The paradoxical ER activity was consistent with expected ER behavior in that it exhibited characteristic binding to the natural ligand, 17 beta-estradiol (E2), and it was inhibited by the antiestrogens, 4-hydroxy-tamoxifen (OHT) and ICI 164384 (ICI). Control vectors containing a truncated antisense ER cDNA produced no ER activity. Although the mechanism for this ER expression has not been determined, it appears likely that it is due to transcription off the opposite strand of the antisense construct.(ABSTRACT TRUNCATED AT 250 WORDS)

Trends among biomedical investigators at top-tier research institutions: a study of the Pew Scholars.

PURPOSE: To gather information and opinions from promising young scientists at top-tier research institutions to learn how they are being affected by the changing biomedical research environment and to present highlights from some of the major reports in the literature on trends in biomedical education and employment in the United States. METHOD: In 1996, the authors conducted a survey of all individuals who had been chosen as awardees in the Pew Scholars Program in the Biomedical Sciences between 1985 and 1995. This group was chosen because it represents independently-identified highly successful investigators at top-tier biomedical research institutions in the United States. RESULTS: Overall, the members of the study group performed better than did their peers nationwide regarding time to degrees, ages at first position, and first awards of federal funding. Nonetheless, even within this above-average cohort, trends were identified that indicate a general aging of young scientists. Not surprisingly, members of this cohort had greater access to federal funds for training and were more likely to pursue careers in academia than their peers nationwide. Despite the success of this well-positioned cohort of scientists, their views on the job market, the supply of biomedical scientists, and the training of students were surprisingly pessimistic. CONCLUSION: The study findings provide information about early career paths of investigators at top-tier research institutions. In addition, the views of this successful cohort serve to inform the current dialogue and questions that remain about the future health of biomedical research and education in the United States. Educators, prospective and current students, and members of the policy community may find it useful to consider these findings and the questions they raise, some of which the authors present.

Characteristics, importance, and implications of comprehensive drug therapy management.

Ideas and opinions about comprehensive drug therapy management (CDTM) were gathered from an expert panel representing health care practitioners, educators, and consumers. A qualitative method of inquiry, the Delphi technique, was used to aggregate opinions of an expert panel of health care professionals, health care educators, and consumer representatives. The 66 experts who agreed to participate in the study consisted of 10 from pharmacy education, 10 from pharmacy professional associations, 20 from managed care, 19 from primary care, and 7 consumers (public members of health care regulatory and governing boards). Each participant was sent a questionnaire designed to evaluate a preliminary definition of CDTM and gather opinions on related topics. A second questionnaire was sent to the participants to determine the extent of their agreement with each statement generated by the first questionnaire. A third questionnaire was sent, asking participants to reconsider their ratings in light of the group's responses and to choose the most important elements. The response rates were 83% for the first questionnaire, 70% for the second, and 76% for the third. The panel reached a high level of agreement on a definition of CDTM. Panel members also identified many critical aspects of CDTM, including what makes it worthwhile, barriers to and facilitators of engaging in CDTM, key participants, ways in which responsibility for CDTM may be shared, and the competencies needed for CDTM. An expert panel of health care leaders and future leaders, within and outside the pharmacy profession, reached a high level of agreement on a definition of CDTM and identified many critical aspects of this concept.

Amino acids bracketing the predicted transmembrane domains of membrane proteins.

The cell membrane is a complex mixture of several classes of biomolecules but amino acids and lipids are the main constituents. For this reason we are establishing a data base of transmembrane proteins with the intent of using the data base to identify interfacial peptide sequences useful for studying protein-lipid interactions at membrane interfaces. Our present intention is to characterize transmembrane peptides and amino acids found near the membrane interface. A data base containing only signal peptides is available (G. von Heijne, Prot. Seq. Data Anal. 1:41-42, 1987).

A novel photoactivatable cross-linker for the functionally-directed region-specific fluorescent labeling of proteins.

A cleavable cross-linking reagent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3'- dithiopropionate (SAED), was synthesized for the selective transfer of a coumarin fluorophore from a 'donor' protein to a position near the binding site of an interacting 'target' protein. SAED contains a terminal N-sulfosuccinimidyl ester for conjugation to the donor, a terminal photoactivatable azido-coumarin species for cross-linking with the interacting target, and a central disulfide spacer for the release of the labeled target after cleavage. To evaluate the effectiveness of this labeling reagent, soybean trypsin inhibitor (STI) was derivatized (approximately 0.5 mol/mol) with SAED and then photolyzed in the presence of trypsin. A single fluorescent cross-linked species (6-7 mol% of total STI) was observed by SDS/PAGE and, after reductive cleavage, was shown to be a 1:1 STI-trypsin complex. This complex was not detected without photolysis or with an inactivated cross-linker. Importantly, complex formation was inhibited by an excess of unmodified STI and prevented by substitution of a non-interacting protein for trypsin. Cleavage of the cross-linked complex revealed that the trypsin, but not the STI, was fluorescent; the uncomplexed trypsin fraction remained unlabeled. These results demonstrated the specificity of the labeling of trypsin by fluorescent-transfer cross-linking with SAED. An efficiency of about 15% for this cross-linking mediated labeling of trypsin was calculated. The short cross-linking span of SAED (less than or equal to 1.8 nm) strictly limited the labeling to the vicinity of the contact region of trypsin with STI. Thus, this novel cross-linker permits the region-specific targeting of a fluorophore near a functionally important binding site.