Bending rigidity of SOPC membranes containing cholesterol.

Bilayer membranes in the fluid state exhibit a large resistance to changes in surface area, negligible resistance to surface shear deformation, and a small but finite resistance to bending. The presence of cholesterol in the membrane is known to increase its resistance to area dilation. In this report, a new method for measuring bilayer membrane bending stiffness has been used to investigate the effect of cholesterol on the bending rigidity of SOPC (1,stearoyl-2,oleoyl-phosphatidylcholine) membranes. The curvature elasticity (kc) for membranes saturated with cholesterol was measured to be 3.3 x 10(-19) J, approximately 3-fold larger than that the modulus for cholesterol-free SOPC membrane. These findings are consistent with previous measurements of bending stiffness based on thermal fluctuations, which showed a similar approximately 3-fold increase in the modulus with cholesterol addition (Evans and Rawicz, 1990, Phys. Rev. Lett. 64:2094) and provide further substantiation of the important contribution that cholesterol makes to membrane cohesion and stability.     

Molecular mapping of genes determining height, time to heading, and growth habit in barley (Hordeum vulgare).

A combination of random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers has been used to locate genes controlling important developmental characters in barley. The denso dwarfing gene has been mapped to the long arm of chromosome 3H. Stepwise multiple regression was also used to identify another region of the barley genome (on chromosome 7H), which contributed to variation in height. The denso locus was shown to be associated with delaying time to heading. A protein (WSP2) and an RAPD marker on barley chromosomes 5H and 6H, respectively, were also associated with time to heading. These results are discussed in relation to the genetic analysis of developmentally important traits and the development of dwarfing genes in barley breeding programs.     

New caerin antibacterial peptides from the skin glands of the Australian tree frog Litoria xanthomera

The secretion of the skin glands of the ‘orange-thighed frog’ Litoria xanthomera contains seven peptides. One of these is the known hypotensive peptide caerulein. Two new peptides, caerin 1.6 [GLFSVLGAVAKHVLPHVVPVIAEKL(NH₂)], and caerin 1.7 [GLFKVLGSVAKHLLPHVAPVIAEKL(NH₂)] show antibacterial properties. Two other peptides lack the first two amino acid residues of caerins 1.6 and 1.7 and show no antibacterial activity. The identification of the peptides in Litoria xanthomera confirms that this species is related to Litoria caerula, Litoria gilleni and Litoria splendida but not as closely as those three species are related to each other. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular Accessibility in Relation to Cell Surface Topography and Compression Against a Flat Substrate

The recruitment of cells to the vascular wall in vivo or the capture of cell subpopulations at the surface of a fabricated device requires the formation of bonds between specific molecular pairs on the cell and the substrate. The ability of a molecule to form a bond depends critically on its localization relative to the cell surface topography. In this report, we present a framework for the quantitative assessment of molecular availability that accounts for the deformability of the cell surface and the balance of forces in the interface, as well as the variability of surface protrusion lengths and the preference for molecules to reside at or away from the tips of surface projections. We also examined how molecular availability should change with increasing compression of the cell against the substrate. Finally, we convolved the distribution of molecules at the interface with a decaying evanescent excitation to predict the fluorescence intensity in total internal reflectance fluorescence microscopy, which can provide a quantitative measure of the relative availability of different molecules at a cell-substrate interface. Model predictions show good agreement with measurements of fluorescence intensity of different molecules labeled fluorescently on the surface of a human neutrophil compressed against a glass surface.     

Cell Surface Topography Is a Regulator of Molecular Interactions during Chemokine-Induced Neutrophil Spreading

Adhesive interactions between neutrophils and endothelium involve chemokine-induced neutrophil spreading and subsequent crawling on the endothelium to sites of transmigration. We investigated the importance of cell topography in this process using immunofluorescence, scanning electron microscopy, and live-cell imaging using total internal reflectance microscopy to observe redistribution of key membrane proteins, both laterally and relative to surface topography, during neutrophil spreading onto glass coated with interleukin 8. During formation of the lamellipod, L-selectin is distributed on microvilli tips along the top of the lamellipodium, whereas the interleukin 8 receptors CXCR1 and CXCR2 and the integrin LFA-1 (α_Lβ₂) were present at the interface between the lamellipodium and the substrate. Total internal reflection fluorescence imaging indicated that LFA-1 and both chemokine receptors redistributed into closer contact with the substrate as the cells spread onto the surface and remodeled their topography. A geometric model of the surface remodeling with nonuniform distribution of molecules and a realistic distribution of microvilli heights was matched to the data, and the fits indicated a 1000-fold increase in the concentration of chemokine receptors and integrins available for bond formation at the interface. These observations imply that topographical remodeling is a key mechanism for regulating cell adhesion and surface-induced activation of cells.     

Comparison of the substrate specificity of two potyvirus proteases

The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate-binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.     

Noncatalytic assembly of ribonuclease III with double-stranded RNA

Ribonuclease III (RNase III) represents a family of double-stranded RNA (dsRNA) endonucleases. The simplest bacterial enzyme contains an endonuclease domain (endoND) and a dsRNA binding domain (dsRBD). RNase III can affect RNA structure and gene expression in either of two ways: as a dsRNA-processing enzyme that cleaves dsRNA, or as a dsRNA binding protein that binds but does not cleave dsRNA. We previously determined the endoND structure of Aquifex aeolicus RNase III (Aa-RNase III) and modeled a catalytic complex of full-length Aa-RNase III with dsRNA. Here, we present the crystal structure of Aa-RNase III in complex with dsRNA, revealing a noncatalytic assembly. The major differences between the two functional forms of RNase III.dsRNA are the conformation of the protein and the orientation and location of dsRNA. The flexibility of a 7 residue linker between the endoND and dsRBD enables the transition between these two forms.     

The structure of Yersinia pestis V-antigen, an essential virulence factor and mediator of immunity against plague

The LcrV protein (V-antigen) is a multifunctional virulence factor in Yersinia pestis, the causative agent of plague. LcrV regulates the translocation of cytotoxic effector proteins from the bacterium into the cytosol of mammalian cells via a type III secretion system, possesses antihost activities of its own, and is also an active and passive mediator of resistance to disease. Although a crystal structure of this protein has been actively sought for better understanding of its role in pathogenesis, the wild-type LcrV was found to be recalcitrant to crystallization. We employed a surface entropy reduction mutagenesis strategy to obtain crystals of LcrV that diffract to 2.2 A and determined its structure. The refined model reveals a dumbbell-like molecule with a novel fold that includes an unexpected coiled-coil motif, and provides a detailed three-dimensional roadmap for exploring structure-function relationships in this essential virulence determinant.     

Elastic properties of the red blood cell membrane that determine echinocyte deformability

The natural biconcave shape of red blood cells (RBC) may be altered by injury or environmental conditions into a spiculated form (echinocyte). An analysis is presented of the effect of such a transformation on the resistance of RBC to entry into capillary sized cylindrical tubes. The analysis accounts for the elasticity of the membrane skeleton in dilation and shear, and the local and nonlocal resistance of the bilayer to bending, the latter corresponding to different area strains in the two leaflets of the bilayer. The shape transformation is assumed to be driven by the equilibrium area difference (A₀, the difference between the equilibrium areas of the bilayer leaflets), which also affects the energy of deformation. The cell shape is approximated by a parametric model. Shape parameters, skeleton shear deformation, and the skeleton density of deformed membrane relative to the skeleton density of undeformed membrane are obtained by minimization of the corresponding thermodynamic potential. Experimentally, A₀ is modified and the corresponding discocyte–echinocyte shape transition obtained by high-pressure aspiration into a narrow pipette, and the deformability of the resulting echinocyte is examined by whole cell aspiration into a larger pipette. We conclude that the deformability of the echinocyte can be accounted for by the mechanical behavior of the normal RBC membrane, where the equilibrium area difference A₀ is modified.