Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

Fitness reduction and potential extinction of wild populations of Atlantic salmon, Salmo salar, as a result of interactions with escaped farm salmon.

The high level of escapes from Atlantic salmon farms, up to two million fishes per year in the North Atlantic, has raised concern about the potential impact on wild populations. We report on a twogeneration experiment examining the estimated lifetime successes, relative to wild natives, of farm, F1 and F2 hybrids and BC1 backcrosses to wild and farm salmon. Offspring of farm and hybrids (i.e. all F1 , F2 and BC1 groups) showed reduced survival compared with wild salmon but grew faster as juveniles and displaced wild parr, which as a group were significantly smaller. Where suitable habitat for these emigrant parr is absent, this competition would result in reduced wild smolt production. In the experimental conditions, where emigrants survived downstream, the relative estimated lifetime success ranged from 2% (farm) to 89% (BC1 wild) of that of wild salmon, indicating additive genetic variation for survival . Wild salmon primarily returned to fresh water after one sea winter (1SW) but farm and hybrids produced proportionately more 2SW salmon. However, lower overall survival means that this would result in reduced recruitment despite increased 2SW fecundity. We thus demonstrate that interaction of farm with wild salmon results in lowered fitness, with repeated escapes causing cumulative fitness depression and potentially an extinction vortex in vulnerable populations.

Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa

Background: Interactions between Mycobacterium avium subsp. paratuberculosis (Map) and free-living protozoa in water are likely to occur in nature. The potential impact of ingestion of Map by two naturally occurring Acanthamoeba spp. on this pathogen's survival and chlorine resistance was investigated. Results: Between 4.6 and 9.1% of spiked populations of three Map strains (NCTC 8578, B2 and ATCC 19698), which had been added at a multiplicity of infection of 10: 1, were ingested by Acanthamoeba castellanii CCAP 1501/1B and A. polyphaga CCAP 1501/3B during co-culture for 3 h at 25 C. Map cells were observed to be present within the vacuoles of the amoebae by acid-fast staining. During extended co-culture of Map NCTC 8578 at 25 degrees C for 24 d with both A. castellanii and A. polyphaga Map numbers did not change significantly during the first 7 days of incubation, however a 1-1.5 log(10) increase in Map numbers was observed between days 7 and 24 within both Acanthamoeba spp. Ingested Map cells were shown to be more resistant to chlorine inactivation than free Map. Exposure to 2 mu g/ml chlorine for 30 min resulted in a log(10) reduction of 0.94 in ingested Map but a log(10) reduction of 1.73 in free Map (p <0.001). Conclusion: This study demonstrated that ingestion of Map by and survival and multiplication of Map within Acanthamoeba spp. is possible, and that Map cells ingested by amoebae are more resistant to inactivation by chlorine than free Map cells. These findings have implications with respect to the efficacy of chlorination applied to Map infected surface waters.

Kinetics of phase transformations in a model with metastable fluid-fluid separation: A molecular dynamics study

By molecular dynamics (MD) simulations we study the crystallization process in a model system whose particles interact by a spherical pair potential with a narrow and deep attractive well adjacent to a hard repulsive core. The phase diagram of the model displays a solid-fluid equilibrium, with a metastable fluid-fluid separation. Our computations are restricted to fairly small systems (from 2592 to 10368 particles) and cover long simulation times, with constant energy trajectories extending up to 76x10(6) MD steps. By progressively reducing the system temperature below the solid-fluid line, we first observe the metastable fluid-fluid separation, occurring readily and almost reversibly upon crossing the corresponding line in the phase diagram. The nucleation of the crystal phase takes place when the system is in the two-fluid metastable region. Analysis of the temperature dependence of the nucleation time allows us to estimate directly the nucleation free energy barrier. The results are compared with the predictions of classical nucleation theory. The critical nucleus is identified, and its structure is found to be predominantly fcc. Following nucleation, the solid phase grows steadily across the system, incorporating a large number of localized and extended defects. We discuss the relaxation processes taking place both during and after the crystallization stage. The relevance of our simulation for the kinetics of protein crystallization under normal experimental conditions is discussed. (C) 2002 American Institute of Physics.

Physicochemical characterization of bioactive polyacrylic acid organogels as potential antimicrobial implants for the buccal cavity

This study describes the formulation and physicochemical characterization of poly(acrylic acid) (PAA) organogels, designed as bioactive implants for improved treatment of infectious diseases of the oral cavity. Organogels were formulated containing a range of concentrations of PAA (3-10% w/w) and metronidazole (2 or 5% w/w, representing a model antimicrobial agent) in different nonaqueous solvents, namely, glycerol (Gly), polyethylene glycol (PEG 400), or propylene glycol (PG). Characterization of the organogels was performed using flow rheometry, compressional analysis, oscillatory rheometry, in vitro mucoadhesion, moisture uptake, and drug release, methods that provide information pertaining to the nonclinical and clinical use of these systems. Increasing the concentration of PAA significantly increased the consistency, compressibility, storage modulus, loss modulus, dynamic viscosity, mucoadhesion, and the rate of drug release. These observations may be accredited to enhanced molecular polymer entanglement. In addition, the choice of solvent directly affected the physicochemical parameters of the organogels, with noticeable differences observed between the three solvents examined. These differences were accredited to the nature of the interaction of PAA with each solvent and, importantly, the density of the resultant physical cross-links. Good correlation was observed between the viscoelastic properties and drug release, with the exception of glycerol-based formulations containing 5 and 10% w/w PAA. This disparity was due to excessive swelling during the dissolution analysis. Ideally, formulations should exhibit controlled drug release, high viscoelasticity, and mucoadhesion, but should flow under minimal stresses. Based on these criteria, PEG 400-based organogels composed of 5% or 10% w/w PAA exhibited suitable physicochemical properties and are suggested to be a potentially interesting strategy for use as bioactive implants designed for use in the oral cavity. © 2008 American Chemical Society.

Unusual voltammetry of the reduction of O-2 in [C(4)dmim][N(Tf)(2)] reveals a strong interaction of O-2(center dot-) with the [C(4)dmim](+) cation

Voltammetric studies of the reduction of oxygen in the room temperature ionic liquid [C(4)dmim][N(Tf)(2)] have revealed a significant positive shift in the back peak potential, relative to that expected for a simple electron transfer. This shift is thought to be due to the strong association of the electrogenerated superoxide anion with the solvent cation. In this work we quantitatively simulate the microdisc electrode voltammetry using a model based upon a one-electron reduction followed by a reversible chemical step, involving the formation of the [C(4)dmim](+)center dot center dot center dot O-2(center dot-) ion-pair, and in doing so we extract a set of parameters completely describing the system. We have simulated the voltammetry in the absence of a following chemical step and have shown that it is impossible to simultaneously fit both the forward and reverse peaks. To further support the parameters extracted from fitting the experimental voltammetry, we have used these parameters to independently simulate the double step chronoamperometric response and found excellent agreement. The parameters used to describe the association of the O-2(center dot-) with the [C(4)dmim](+) were k(f) = 1.4 x 10(3) s(-1) for the first-order rate constant and K-eq = 25 for the equilibrium constant.

Radiation-induced bystander and adaptive responses in cell and tissue models.

The use of microbeam approaches has been a major advance in probing the relevance of bystander and adaptive responses in cell and tissue models. Our own studies at the Gray Cancer Institute have used both a charged particle microbeam, producing protons and helium ions and a soft X-ray microprobe, delivering focused carbon-K, aluminium-K and titanium-K soft X-rays. Using these techniques we have been able to build up a comprehensive picture of the underlying differences between bystander responses and direct effects in cell and tissue-like models. What is now clear is that bystander dose-response relationships, the underlying mechanisms of action and the targets involved are not the same as those observed for direct irradiation of DNA in the nucleus. Our recent studies have shown bystander responses even when radiation is deposited away from the nucleus in cytoplasmic targets. Also the interaction between bystander and adaptive responses may be a complex one related to dose, number of cells targeted and time interval.

Multimodal Biometric Human Recognition for Perceptual Human-Computer Interaction

In this paper, a novel video-based multimodal biometric verification scheme using the subspace-based low-level feature fusion of face and speech is developed for specific speaker recognition for perceptual human--computer interaction (HCI). In the proposed scheme, human face is tracked and face pose is estimated to weight the detected facelike regions in successive frames, where ill-posed faces and false-positive detections are assigned with lower credit to enhance the accuracy. In the audio modality, mel-frequency cepstral coefficients are extracted for voice-based biometric verification. In the fusion step, features from both modalities are projected into nonlinear Laplacian Eigenmap subspace for multimodal speaker recognition and combined at low level. The proposed approach is tested on the video database of ten human subjects, and the results show that the proposed scheme can attain better accuracy in comparison with the conventional multimodal fusion using latent semantic analysis as well as the single-modality verifications. The experiment on MATLAB shows the potential of the proposed scheme to attain the real-time performance for perceptual HCI applications.

Application of the zero-range potential model to positron annihilation on molecules

In this paper we use a zero-range potential (ZRP) method to model positron interaction with molecules. This allows us to investigate the e?ect of molecular vibrations on positron–molecule annihilation using the van der Waals dimer Kr2 as an example. We also use the ZRP to explore positron binding to polyatomics and examine the dependence of the binding energy on the size of the molecule for alkanes. We ?nd that a second bound state appears for a molecule with ten carbons, similar to recent experimental evidence for such a state emerging in alkanes with twelve carbons.

Electron beam-plasma interaction and ion-acoustic solitary waves in plasmas with a superthermal electron component

The study of non-Maxwellian plasmas is crucial to the understanding of space and astrophysical plasma dynamics. In this paper, we investigate the existence of arbitrary amplitude ion-acoustic solitary waves in an unmagnetized plasma consisting of ions and excess superthermal electrons (modelled by a kappa-type distribution), which is penetrated by an electron beam. A kappa (kappa-) type distribution is assumed for the background electrons. A (Sagdeev-type) pseudopotential formalism is employed to derive an energy-balance like equation. The range of allowed values of the soliton speed (Mach number), wherein solitary waves may exist, is determined. The Mach number range (allowed soliton speed values) becomes narrower under the combined effect of the electron beam and of the superthermal electrons, and may even be reduced to nil (predicting no solitary wave existence) for high enough beam density and low enough kappa (significant superthermality). For fixed values of all other parameters (Mach number, electron beam-to-ion density ratio and electron beam velocity), both soliton amplitude and (electric potential perturbation) profile steepness increase as kappa decreases. The combined occurrence of small-amplitude negative potential structures and larger amplitude positive ones is pointed out, while the dependence of either type on the plasma parameters is investigated.

Outgrowth Endothelial Cells: Characterization and Their Potential for Reversing Ischemic Retinopathy

Purpose. Endothelial progenitor cells (EPCs) have potential for promoting vascular repair and revascularization of ischemic retina. However, the highly heterogeneous nature of these cells causes confusion when assessing their biological functions. The purpose of this study was to provide a comprehensive comparison between the two main EPC subtypes, early EPCs (eEPCs) and outgrowth endothelial cells (OECs), and to establish the potential of OECs as a novel cell therapy for ischemic retinopathy.

Methods. Two types of human blood-derived EPCs were isolated and compared using immunophenotyping and multiple in vitro functional assays to assess interaction with retinal capillary endothelial cells and angiogenic activity. OECs were delivered intravitreally in a mouse model of ischemic retinopathy, and flat mounted retinas were examined using confocal microscopy.

Results. These data indicate that eEPCs are hematopoietic cells with minimal proliferative capacity that lack tube-forming capacity. By contrast, OECs are committed to an endothelial lineage and have significant proliferative and de novo tubulogenic potential. Furthermore, only OECs are able to closely interact with endothelial cells through adherens and tight junctions and to integrate into retinal vascular networks in vitro. The authors subsequently chose OECs to test a novel cell therapy approach for ischemic retinopathy. Using a murine model of retinal ischemia, they demonstrated that OECs directly incorporate into the resident vasculature, significantly decreasing avascular areas, concomitantly increasing normovascular areas, and preventing pathologic preretinal neovascularization.

Conclusions. As a distinct EPC population, OECs have potential as therapeutic cells to vascularize the ischemic retina.

Attosecond phase locking of harmonics emitted from laser-produced plasmas

Laser-driven coherent extreme-ultraviolet (XUV) sources provide pulses lasting a few hundred attoseconds(1,2), enabling real-time access to dynamic changes of the electronic structure of matter(3,4), the fastest processes outside the atomic nucleus. These pulses, however, are typically rather weak. Exploiting the ultrahigh brilliance of accelerator-based XUV sources(5) and the unique time structure of their laser-based counterparts would open intriguing opportunities in ultrafast X-ray and high-field science, extending powerful nonlinear optical and pump-probe techniques towards X-ray frequencies, and paving the way towards unequalled radiation intensities. Relativistic laser-plasma interactions have been identified as a promising approach to achieve this goal(6-13). Recent experiments confirmed that relativistically driven overdense plasmas are able to convert infrared laser light into harmonic XUV radiation with unparalleled efficiency, and demonstrated the scalability of the generation technique towards hard X-rays(14-19). Here we show that the phases of the XUV harmonics emanating from the interaction processes are synchronized, and therefore enable attosecond temporal bunching. Along with the previous findings concerning energy conversion and recent advances in high-power laser technology, our experiment demonstrates the feasibility of confining unprecedented amounts of light energy to within less than one femtosecond.

The GAL genetic switch: visualisation of the interacting proteins by split-EGFP bimolecular fluorescence complementation

A split-EGFP bimolecular fluorescence complementation assay was used to visualise and locate three interacting pairs of proteins from the GAL genetic switch of the budding yeast, Saccharomyces cerevisiae. Both the Gal4p-Gal80p and Gal80p-Gal3p pairs were found to be located in the nucleus under inducing conditions. However, the Gal80p-Gal1p complex was located throughout the cell. These results support recent work establishing an initial interaction between Gal3p and Gal80p occurring in the nucleus. Labelling of all three protein pairs impaired the growth of the yeast strains and resulted in reduced galactokinase activity in cell extracts. The most likely cause of this impairment is decreased dissociation rates of the complexes, caused by the essentially irreversible reassembly of the EGFP fragments. This suggests that a fully functional GAL genetic switch requires dynamic interactions between the protein components. These results also highlight the need for caution in the interpretation of in vivo split-EGFP experiments.

The synthesis and electronic structure of a novel [NiS4Fe2(CO)(6)] radical cluster: Implications for the active site of the [NiFe] hydrogenases

A novel [Ni'S-4'Fe-2(CO)(6)] cluster (1: 'S-4'=(CH3C6H3S2)(2)(CH2)(3)) has been synthesised, structurally characterised and has been shown to undergo a chemically reversible reduction process at -1.31 V versus Fc(+)/Fc to generate the EPR-active monoanion 1(-). Multifrequency Q-, X- and S-band EPR spectra of Ni-61-enriched 1(-) show a well-resolved quartet hyperfine splitting in the low-field region due to the interaction with a single Ni-61 (I = 3/2) nucleus. Simulations of the EPR spectra require the introduction of a single angle of non-coincidence between g, and A(1), and g(3) and A(3) to reproduce all of the features in the S- and X-band spectra. This behaviour provides a rare example of the detection and measurement of non-coincidence effects from frozen-solution EPR spectra without the need for single-crystal measurements, and in which the S-band experiment is sensitive to the non-coincidence. An analysis of the EPR spectra of 1(-) reveals a 24% Ni contribution to the SOMO in 1(-), supporting a delocalisation of the spin-density across the NiFe2 cluster. This observation is supported by IR spectroscopic results which show that the CO stretching frequencies, v(CO), shift to lower frequency by about 70 cm(-1) when 1 is reduced to 1(-). Density functional calculations provide a framework for the interpretation of the spectroscopic properties of 1(-) and suggest that the SOMO is delocalised over the whole cluster, but with little S-centre participation. This electronic structure contrasts with that of the Ni-A, -B, -C and -L forms of [NiFe] hydrogenase in which there is considerable S participation in the SOMO.

FASCIOLA-HEPATICA - THE EFFECT OF THE MICROTUBULE INHIBITORS COLCHICINE AND TUBULOZOLE-C ON THE ULTRASTRUCTURE OF THE ADULT FLUKE

The effect of the microtubule inhibitors colchicine (1 x 10(-3) M) and tubulozole-C(1 x 10(-6) M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of Tl secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type 1 cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, 'yolk' globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.