Mutational analysis of the membrane proximal heptad repeat of the newcastle disease virus fusion protein.

Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, that have been implicated in the fusion activity of the protein. Peptides from these two domains form a six-stranded, coiled-coil with the HR1 sequences forming a central trimer and three molecules of the HR2 helix located within the grooves in the central trimer (Baker et al., 1999, Mol. Cell 3, 309; Zhao et al. 2000, Proc. Natl. Acad. Sci. USA 97, 14172). Nonconservative mutations were made in the HR2 domain of the Newcastle disease virus fusion protein in residues that are likely to form contacts with the HR1 core trimer. These residues form the hydrophobic face of the helix and adjacent residues ("a" and "g" positions in the HR2 helical wheel structure). Mutant proteins were characterized for effects on synthesis, steady-state levels, proteolytic cleavage, and surface expression as well as fusion activity as measured by syncytia formation, content mixing, and lipid mixing. While all mutant proteins were transport competent and proteolytically cleaved, these mutations did variously affect fusion activity of the protein. Nonconservative mutations in the "g" position had no effect on fusion. In contrast, single changes in the middle "a" position of HR2 inhibited lipid mixing, content mixing, and syncytia formation. A single mutation in the more carboxyl-terminal "a" position had minimal effects on lipid mixing but did inhibit content mixing and syncytia formation. These results are consistent with the idea that the HR2 domain is involved in posttranslational interactions with HR1 that mediate the close approach of membranes. These results also suggest that the HR2 domain, particularly the carboxyl-terminal region, plays an additional role in fusion, a role related to content mixing and syncytia formation.

Analogical versus rule-based classification.

Sensitivity to feature co-occurrences was investigated as a function of analytic and analogical transfer. Participants memorized descriptions of hypothetical people and were then induced either to make transfer decisions by analogy to the descriptions (analogical transfer) or to search for and apply rules (analytic transfer). Across three experiments, analogical transfer was found to be more effective than analytic transfer for preserving co-occurring features in classification judgements. This result held for a variety of category structures and stimulus materials. It was difficult for subjects who adopted an analytic-transfer strategy to identify regularities that were embedded in stored instances. Alternatively, subjects who adopted an analogical-transfer strategy preserved feature co-occurrences as an indirect result of similarity-based retrieval and comparison processes. The effectiveness of analytic and analogical transfer is discussed.

Analytical procedure for the determination of the new antitumour drug N-benzoylstaurosporine and three potential metabolites in human plasma by reversed-phase high-performance liquid chromatography.

The staurosporine derivative, N-benzoylstaurosporine (CGP 41 251; I), is a protein kinase C inhibitor that has been selected for phase I clinical evaluation in cancer patients. We have developed a selective and sensitive assay of the drug and three potential metabolites in human plasma. The method is based on reversed-phase high-performance liquid chromatography with fluorescence detection. The sample pretreatment involves liquid-liquid extraction with diisopropyl ether with recoveries over 88%. The limit of detection and limit of quantitation of the parent compound and two metabolites were 0.5 and 1.0 ng/ml, respectively. For the third metabolite the limit of detection and limit of quantitation were 1.0 and 2.0 ng/ml, respectively. Linear calibration lines were obtained over the range of 1-1000 ng/ml. The between-day and within-day precisions were < 7.1% for all the analytes. In plasma the compounds were stable for at least one month if stored at -30 degrees C or below. The applicability of the method for in vivo studies has been demonstrated in a pharmacokinetic study in rat receiving 0.5 mg/kg of the drug as an intravenous bolus injection. Compound I and two metabolites were detected.