Differences in specificity and catalytic efficiency between allozymes of esterase-4 from Drosophila mojavensis

A more than 10-fold difference in the specificity and catalytic efficiency for 1-naphthyl esters was measured between two allozymes of esterase-4 from Drosophila mojavensis. This difference is mainly caused by a difference in the affinity for the 1-naphthyl esters. The amino acid compositions of the allozymes are not significantly different, which means that the difference in primary structure is small. Small differences in primary structure generally do not result in such a large increase in catalytic efficiency and such a large shift in substrate specificity as was found in the present study.      

Reprogramming homing endonuclease specificity through computational design and directed evolution

Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally.     

Electron microscopic study of the role of lipid micelles in intestinal fat absorption

In vitro micellar solutions of oleic acid, monoolein, and sodium taurocholate were studied electron microscopically. They contained osmiophilic particles 30-200A in diameter. Osmium staining alone was sufficient to demonstrate the particles; lead staining had little effect on their appearance. The intestinal intraluminal contents from rats during the absorption of unsaturated fat also contained osmiophilic particles, 40-200A, and numerous similar particles were found between the microvilli and engaged in the fine filamentous coating of microvilli. In the lumen only, larger emulsion-type droplets were also seen. The small particles were demonstrable in osmium-fixed material, with or without lead stains, and staining with lead only increased contrast of the particles. Spherules measuring about 1000A in diameter with walls about 100A thick were observed in the terminal web during fat absorption, at which time they were slightly larger and more numerous than in fasting rats. It is proposed that during fat absorption micellar particles are engaged in the filamentous material covering the microvilli and then enter the absorptive cell either by molecular diffusion across the plasma membrane or by being incorporated into the walls of thick-coated spherules which then pass into the subapical cytoplasm.