T-loop assembly in vitro involves binding of TRF2 near the 3' telomeric overhang

Mammalian telomeres contain a duplex TTAGGG-repeat tract terminating in a 3' single-stranded overhang. TRF2 protein has been implicated in remodeling telomeres into duplex lariats, termed t-loops, in vitro and t-loops have been isolated from cells in vivo. To examine the features of the telomeric DNA essential for TRF2-promoted looping, model templates containing a 500 bp double-stranded TTAGGG tract and ending in different single-stranded overhangs were constructed. As assayed by electron microscopy, looped molecules containing most of the telomeric tract are observed with TRF2 at the loop junction. A TTAGGG-3' overhang of at least six nucleotides is required for loop formation. Termini with 5' overhangs, blunt ends or 3' termini with non-telomeric sequences at the junction are deficient in loop formation. Addition of non-telomeric sequences to the distal portion of a 3' overhang beginning with TTAGGG repeats only modestly diminishes looping. TRF2 preferentially localizes to the junction between the duplex repeats and the single-stranded overhang. Based on these findings we suggest a model for the mechanism by which TRF2 remodels telomeres into t-loops.     

Structure-function relationships in heparin cofactor II: chemical modification of arginine and tryptophan and demonstration of a two-domain structure

Heparin cofactor II and antithrombin III are plasma proteins functionally similar in their ability to inhibit thrombin at accelerated rates in the presence of heparin. To further characterize the structural and functional properties of human heparin cofactor II as compared to antithrombin III, we studied the possible significance of arginyl and tryptophanyl residues and the changes in protein structure and activity during guanidinium chloride (GdmCl) denaturation. Both antithrombin and heparin cofactor activities of heparin cofactor II are inactivated by the arginine-specific reagent, 2,3-butanedione. Saturation kinetics are observed during modification and suggest formation of a reversible protease inhibitor-butanedione complex. Quantitation of arginyl residues following butanedione modification shows a loss of about four residues for total inactivation, one of which is essential for antithrombin activity. Arginine-modified heparin cofactor II did not bind to heparin-agarose and implies a role for the other modified arginyl residues during heparin cofactor activity. N-Bromosuccinimide oxidation (20 mol of reagent/mol of protein) of heparin cofactor II results in modification of approximately two tryptophanyl residues with no concomitant loss of heparin cofactor activity. Moreover, there is no enhancement of intrinsic protein fluorescence during heparin binding to the native inhibitor. Circular dichroism measurements show that the structural transition of heparin cofactor II during denaturation is distinctly biphasic, yielding midpoints at 0.6 and 2.6 M GdmCl. Functional protease inhibitory activities are affected to the same extent following denaturation-renaturation at various GdmCl concentrations. The results indicate that arginyl residues are critical for both antithrombin and heparin binding activities. In contrast, tryptophanyl residues are apparently not essential for heparin-dependent interactions. The results also suggest that heparin cofactor II contains two structural domains which unfold at different GdmCl concentrations.     

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.     

Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins

We have used low shear viscometry and electron microscopy to study the interaction between pure actin filaments and microtubules. Mixtures of microtubules having microtubule-associated proteins (MAPs) with actin filament have very high viscosities compared with the viscosities of the separate components. MAPs themselves also cause a large increase in the viscosity of actin filaments. In contrast, mixtures of actin filaments with tubulin polymers lacking MAPs have low viscosities, close to the sum of the viscosities of the separate components. Our interpretation of these observations is that there is an interaction between actin filaments and microtubules which requires MAPs. This interaction is inhibited by ATP and some related compounds. Electron micrographs of thin sections through mixtures of actin and microtubules show numerous close associations between the two polymers which may be responsible for their high viscosity.     

The molecular reorganization of lipid bilayers by osmium tetroxide. A spin-label study of orientation and restricted y-axis anisotropic motion in model membrane systems

Phospholipid dispersions spontaneously form oriented lamellar multilayers when dried onto glass slides. These oriented multilayers form useful model systems for studying the molecular dynamics of lipid bilayers. In order to examine the effects of osmium tetroxide on the orientation and motion of hydrocarbon chains in lipid bilayers, lecithin multilayers containing the spin label 3-doxyl-5α-cholestane (the 4′,4′-dimethyloxazolidine-N-oxyl derivative of 5α-cholestan-3-one) were prepared and examined by electron spin resonance spectroscopy. In egg lecithin multilayers at room temperature and 81% relative humidity the osmium tetroxide causes nearly complete loss of orientation and severe reduction of molecular motion. In contrast, the high degree of order in l-α-dipalmitoyl lecithin multilayers is not affected by exposure to osmium tetroxide vapors. Experiments are also reported on macroscopically disordered lecithin preparations, and the data support the conclusions drawn from the ordered lecithin multilayers that rotational mobility of the probe is severely reduced by fixation of the lipid chains.A simple mathematical model has been developed to account for the amplitude of the high-frequency (τ < 10^?8 sec) restricted y-axis anisotropic motion occurring in the bilayer plane. Since the y-axis is roughly parallel to the molecular axis of the rigid steroid spin label, this model enables quantitative comparisons of various degrees of restricted motion about the molecular axis.     

Aerenchymatous phellem in hypocotyl and roots enables O2 transport in Melilotus siculus

? Aerenchymatous phellem (secondary aerenchyma) has rarely been studied in roots. Its formation and role in internal aeration were evaluated for Melilotus siculus, an annual legume of wet saline land. ? Plants were grown for 21 d in aerated or stagnant (deoxygenated) agar solutions. Root porosity and maximum diameters were measured after 0, 7, 14 and 21 d of treatment. Phellem anatomy was studied and oxygen (O(2)) transport properties examined using methylene blue dye and root-sleeving O(2) electrodes. ? Interconnecting aerenchymatous phellem developed in hypocotyl, tap root and older laterals (but not in aerial shoots), with radial intercellular connections to steles. Porosity of main roots containing phellem was c. 25%; cross-sectional areas of this phellem were threefold greater for stagnant than for aerated treatments. Root radial O(2) loss was significantly reduced by complete hypocotyl submergence; values approached zero after disruption of hypocotyl phellem below the waterline or, after shoot excision, by covering hypocotyl phellem in nontoxic cream. ? Aerenchymatous phellem enables hypocotyl-to-root O(2) transport in M. siculus. Phellem increases radially under stagnant conditions, and will contribute to waterlogging tolerance by enhancing root aeration. It seems likely that with hypocotyl submerged, O(2) will diffuse via surface gas-films and internally from the shoot system.     

The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments

The bacteriophage T4 uvsX gene codes for a DNA-binding protein that is important for genetic recombination in T4-infected cells. This protein is a DNA-dependent ATPase that resembles the Escherichia coli recA protein in many of its properties. We have examined the binding of purified uvsX protein to single-stranded DNA (ssDNA) and to double-stranded DNA (dsDNA) using electron microscopy to visualize the complexes that are formed and double label analysis to measure their protein content. We find that the uvsX protein binds cooperatively to dsDNA, forming filaments 14 nm in diameter with an apparently helical axial repeat of 12 nm. Each repeat contains about 42 base pairs and 9-12 uvsX protein monomers. In solutions containing Mg2+, the uvsX protein also binds cooperatively to ssDNA. The filaments that result are 14 nm in diameter, show a 12-nm axial repeat, and they are nearly identical in appearance to the filaments that contain dsDNA. In the filaments formed along ssDNA, each axial repeat contains about 49 DNA bases and 9-12 uvsX monomers. Both the filaments formed on the ssDNA and dsDNA show a strong tendency to align side-by-side. T4 gene 32 protein also binds cooperatively to ssDNA and interacts both physically and functionally with uvsX protein. However, when gene 32 and uvsX proteins were added to ssDNA together, no interaction between the two proteins was detected.     

When mothers make sons sexy: maternal effects contribute to the increased sexual attractiveness of extra-pair offspring

Quality differences between offspring sired by the social and by an extra-pair partner are usually assumed to have a genetic basis, reflecting genetic benefits of female extra-pair mate choice. In the zebra finch (Taeniopygia guttata), we identified a colour ornament that is under sexual selection and appears to have a heritable basis. Hence, by engaging in extra-pair copulations with highly ornamented males, females could, in theory, obtain genes for increased offspring attractiveness. Indeed, sons sired by extra-pair partners had larger ornaments, seemingly supporting the genetic benefit hypothesis. Yet, when comparing ornament size of the social and extra-pair partners, there was no difference. Hence, the observed differences most likely had an environmental basis, mediated, for example, via differential maternal investment of resources into the eggs fertilized by extra-pair and social partners. Such maternal effects may (at least partly) be mediated by egg size, which we found to be associated with mean ornament expression in sons. Our results are consistent with the idea that maternal effects can shape sexual selection by altering the genotype-phenotype relationship for ornamentation. They also caution against automatically attributing greater offspring attractiveness or viability to an extra-pair mate's superior genetic quality, as without controlling for differential maternal investment we may significantly overestimate the role of genetic benefits in the evolution of extra-pair mating behaviour.     

Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05⁺ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05⁺ NK cells, including three immunodominant CD8⁺ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05⁺ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002⁺ CD4⁺ lymphocytes co-cultured with Mamu-KIR3DL05⁺ NK cells than with Mamu-KIR3DL05^- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses.