Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors

Mouse embryos at the 2-cell stage were cultured in the presence of cytochalasin B (CB), cytochalasin D (CD), colchicine (COL) or colcemid (COM) for up to 72 h. Cleavage was arrested in the 2-cell and 8-cell embryos cultured in CB or CD but the blastomeres continued to differentiate, since chromosome replication occurred in the blastomeres at approximately the same time as control embryos underwent cleavage; an increase in the incorporation of [³H]uridine into RNA was also detected. Furthermore, the cleavage-arrested embryos acquired the necessary information to undergo morphogenesis; these embryos when explanted to fresh medium after 48 h culture in CB or CD underwent compaction within 15–60 min and started to cavitate to produce trophoblastic vesicles within 5–6 h at the same time as when the control embryos were undergoing compaction and beginning to form blastocoelic cavities. In contrast, the embryos arrested in the presence of COM or COL showed none of these differentiative, biochemical or morphogenetic changes. Hence, differentiation of blastomeres and morphogenesis is apparently coupled with nuclear divisions and the information does not reside within the blastomeres at the 2-cell or 8-cell stage. The trophoblastic vesicles produced after cleavage arrest subsequently gave rise to only trophoblast giant cells and no embryonic derivatives were detected.     

Inhibition of Human Immunodeficiency Virus Type 1 Infectivity by the gp41 Core: Role of a Conserved Hydrophobic Cavity in Membrane Fusion

The gp41 envelope protein of human immunodeficiency virus type 1 (HIV-1) contains an alpha-helical core structure responsible for mediating membrane fusion during viral entry. Recent studies suggest that a conserved hydrophobic cavity in the coiled coil of this core plays a distinctive structural role in maintaining the fusogenic conformation of the gp41 molecule. Here we investigated the importance of this cavity in determining the structure and biological activity of the gp41 core by using the N34(L6)C28 model. The high-resolution crystal structures of N34(L6)C28 of two HIV-1 gp41 fusion-defective mutants reveal that each mutant sequence is accommodated in the six-helix bundle structure by forming the cavity with different sets of atoms. Remarkably, the mutant N34(L6)C28 cores are highly effective inhibitors of HIV-1 infection, with 5- to 16-fold greater activity than the wild-type molecule. The enhanced inhibitory activity by fusion-defective mutations correlates with local structural perturbations close to the cavity that destabilize the six-helix bundle. Taken together, these results indicate that the conserved hydrophobic coiled-coil cavity in the gp41 core is critical for HIV-1 entry and its inhibition and provides a potential antiviral drug target.