Protein 4.1R (4.1R) is a multifunctional component of the red cell membrane. It forms a ternary complex with actin and spectrin, which defines the nodal junctions of the membrane-skeletal network, and its attachment to the transmembrane protein glycophorin C creates a bridge between the protein network and the membrane bilayer. We now show that deletion of 4.1R in mouse red cells leads to a large diminution of actin accompanied by extensive loss of cytoskeletal lattice structure, with formation of bare areas of membrane. Whereas band 3, the preponderant transmembrane constituent, and proteins known to be associated with it are present in normal or increased amounts, glycophorin C is missing and XK, Duffy, and Rh are much reduced in the 4.1R-deficient cells. The inference that these are associated with 4.1R was borne out by the results of in vitro pulldown assays. Furthermore, whereas Western blot analysis showed normal levels of band 3 and Kell, flow cytometric analysis using an antibody against the extracellular region of band 3 or Kell revealed reduction of these two proteins, suggesting a conformational change of band 3 and Kell epitopes. Taken together, we suggest that 4.1R organizes a macromolecular complex of skeletal and transmembrane proteins at the junctional node and that perturbation of this macromolecular complex not only is responsible for the well characterized membrane instability but may also remodel the red cell surface. macromolecular complex ͉ cytoskeleton

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T m ) ranging from 21 to 72°C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol ؊1 . Constructs comprising five successive repeats from the ␤-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation.

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The change in cell surface properties in the presence of electric currents is of critical concern when the potential to manipulate bacterial movement with electric fields is evaluated. In this study, the effects of different direct electric currents on the cell surface properties involved in bacterial adhesion were investigated by using a mixed phenol-degrading bacterial culture in the exponential growth phase. The traits investigated were surface hydrophobicity (measured by adherence to n-octane), net surface electrostatic charge (determined by measurement of the zeta potential), and the cell surface shape and polymers (determined by scanning electron microscope analysis). The results showed that a lower current (less than 20 mA) induced no significant changes in the surface properties of phenol-degrading bacteria, that an electric current of 20 mA could increase the surface hydrophobicity and flatten the cell shape, and that a higher current (40 mA) could increase the surface extracellular substances and the net negative surface electrostatic charge. The results also revealed that the electric current effects on cell hydrophobicity varied with the suspending medium. We suggest that an electric current greater than 20 mA is not suitable for use in manipulation of the movement of the phenol-degrading bacteria, although such a current might favor the electrophoretic movement of the bacterial species.

BackgroundThe C-terminal domain (CTD) of HIV-1 capsid (CA), like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation. Mutations of the residues at the CTD dimer interface impair virus assembly and render the virus non-infectious. Therefore, the CTD represents a potential target for designing anti-HIV-1 drugs.ResultsDue to the pivotal role of the dimer interface, we reasoned that peptides from the α-helical region of the dimer interface might be effective as decoys to prevent CTD dimer formation. However, these small peptides do not have any structure in solution and they do not penetrate cells. Therefore, we used the hydrocarbon stapling technique to stabilize the α-helical structure and confirmed by confocal microscopy that this modification also made these peptides cell-penetrating. We also confirmed by using isothermal titration calorimetry (ITC), sedimentation equilibrium and NMR that these peptides indeed disrupt dimer formation. In in vitro assembly assays, the peptides inhibited mature-like virus particle formation and specifically inhibited HIV-1 production in cell-based assays. These peptides also showed potent antiviral activity against a large panel of laboratory-adapted and primary isolates, including viral strains resistant to inhibitors of reverse transcriptase and protease.ConclusionsThese preliminary data serve as the foundation for designing small, stable, α-helical peptides and small-molecule inhibitors targeted against the CTD dimer interface. The observation that relatively weak CA binders, such as NYAD-201 and NYAD-202, showed specificity and are able to disrupt the CTD dimer is encouraging for further exploration of a much broader class of antiviral compounds targeting CA. We cannot exclude the possibility that the CA-based peptides described here could elicit additional effects on virus replication not directly linked to their ability to bind CA-CTD.

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KdpFABC is an ATP-dependent K+ pump that ensures bacterial survival in K+-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down regulation when K+ levels are restored has not been described. Here we show that KdpFABC is inhibited when cells return to a K+-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K+-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.

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