Beta-lactoglobulin fibers under capillary flow

We describe the capillary flow behavior of gels of beta-lactoglobulin (beta-lg) containing droplets of fibrils and the shear flow alignment of beta-lg fibers in dilute aqueous solutions. Polarized optical microscopy and laser scanning confocal microscopy are used to show that capillary shear flow does not affect the fibril droplet sizes in the beta-lg gels, the system behaving in this respect as a solution of compact colloidal particles under shear flow. Small-angle X-ray scattering (SAXS) on dilute aqueous solutions indicates that the fibers can be initially aligned under capillary shear, but this alignment is lost after 18 min of shear. Transmission electron microscopy experiments on the samples studied by SAXS suggest that the loss of orientation is due to a shear-induced breakup of the swollen fibril network. Dynamic and static light scattering on dilute beta-lg fibril aqueous solutions are used to show that before shear beta-lg fibrils behave as strongly interacting semiflexible polymers, while they behave as weakly interacting rods after 18 min of capillary shear.

PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E-3-ligase MIR

PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome. PDGF induced Src-dependent association between the multifunctional transcription/translation regulator hnRNP-K and the mRNA-encoding myosin regulatory light-chain (MRLC)-interacting protein (MIR), a E3-ubiquitin ligase that is MRLC specific. This in turn rapidly increased MIR expression, and led to ubiquitination and proteasome-mediated degradation of MRLC. Downregulation of MIR by RNA muting prevented the reorganization of actin structures and severely reduced the migratory and wound-healing potential of PDGF-treated cells. The results show that activation of MIR and the resulting removal of diphosphorylated MRLC are essential for PDGF to instigate and maintain control over the actin-myosin-based contractile system in Swiss 3T3 fibroblasts. The PDGF induced protein destabilization through the regulation of hnRNP-K controlled ubiquitin-ligase translation identifies a novel pathway by which external stimuli can regulate phenotypic development through rapid, organelle-specific changes in the activity and stability of cytoskeletal regulators.

Regulation of actin assembly by SCAR/WAVE proteins.

Actin reorganization is a tightly regulated process that co-ordinates complex cellular events, such as cell migration, chemotaxis, phagocytosis and adhesion, but the molecular mechanisms that underlie these processes are not well understood. SCAR (suppressor of cAMP receptor)/WAVE [WASP (Wiskott-Aldrich syndrome protein)-family verprolin homology protein] proteins are members of the conserved WASP family of cytoskeletal regulators, which play a critical role in actin dynamics by triggering Arp2/3 (actin-related protein 2/3)-dependent actin nucleation. SCAR/WAVEs are thought to be regulated by a pentameric complex which also contains Abi (Abl-interactor), Nap (Nck-associated protein), PIR121 (p53-inducible mRNA 121) and HSPC300 (haematopoietic stem progenitor cell 300), but the structural organization of the complex and the contribution of its individual components to the regulation of SCAR/WAVE function remain unclear. Additional features of SCAR/WAVE regulation are highlighted by the discovery of other interactors and distinct complexes. It is likely that the combinatorial assembly of different components of SCAR/WAVE complexes will prove to be vital for their roles at the centre of dynamic actin reorganization.

Electric field alignment in thin films of cylinder-forming diblock copolymer

We investigate thin films of cylinder-forming diblock copolymer confined between electrically charged parallel plates, using self-consistent-field theory ( SCFT) combined with an exact treatment for linear dielectric materials. Our study focuses on the competition between the surface interactions, which tend to orient cylinder domains parallel to the plates, and the electric field, which favors a perpendicular orientation. The effect of the electric field on the relative stability of the competing morphologies is demonstrated with equilibrium phase diagrams, calculated with the aid of a weak-field approximation. As hoped, modest electric fields are shown to have a significant stabilizing effect on perpendicular cylinders, particularly for thicker films. Our improved SCFT-based treatment removes most of the approximations implemented by previous approaches, thereby managing to resolve outstanding qualitative inconsistencies among different approximation schemes.

Factors governing cellular convection in orographic precipitation

The development of shallow cellular convection in warm orographic clouds is investigated through idealized numerical simulations of moist flow over topography using a cloud-resolving numerical model. Buoyant instability, a necessary element for moist convection, is found to be diagnosed most accurately through analysis of the moist Brunt–Väisälä frequency (N_m) rather than the vertical profile of θ_e. In statically unstable orographic clouds (N_m^2) < 0), additional environmental and terrain-related factors are shown to have major effects on the amount of cellularity that occurs in 2D simulations. One of these factors, the basic-state wind shear, may suppress convection in 2D yet allow for longitudinal convective roll circulations in 3D. The presence of convective structures within an orographic cloud substantially enhanced the maximum rainfall rates, precipitation efficiencies, and precipitation accumulations in all simulations.

Metal bioaccumulation and cellular fractionation in an epigeic earthworm (Lumbricus rubellus): the interactive influences of population exposure histories, site-specific geochemistry and mitochondrial genotype

Subcellular fractionation techniques were used to describe temporal changes (at intervals from T0 to T70 days) in the Pb, Zn and P partitioning profiles of Lumbricus rubellus populations from one calcareous (MDH) and one acidic (MCS) geographically isolated Pb/Zn-mine sites and one reference site (CPF). MDH and MCS individuals were laboratory maintained on their native field soils; CPF worms were exposed to both MDH and MCS soils. Site-specific differences in metal partitioning were found: notably, the putatively metal-adapted populations, MDH and MCS, preferentially partitioned higher proportions of their accumulated tissue metal burdens into insoluble CaPO4-rich organelles compared with naive counterparts, CPF. Thus, it is plausible that efficient metal immobilization is a phenotypic trait characterising metal tolerant ecotypes. Mitochondrial cytochrome oxidase II (COII) genotyping revealed that the populations indigenous to mine and reference soils belong to distinct genetic lineages, differentiated by 13%, with 7 haplotypes within the reference site lineage but fewer (3 and 4, respectively) in the lineage common to the two mine sites. Collectively, these observations raise the possibility that site-related genotype differences could influence the toxico-availability of metals and, thus, represent a potential confounding variable in field-based eco-toxicological assessments.

Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing similar to600 mg of either c9,t11 CIA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dosedependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CIA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.

Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice

Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear: cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice. (C) 2009 Elsevier B.V. All rights reserved.

S-nitrosylation of proteins at the leading edge of migrating trophoblasts by inducible nitric oxide synthase promotes trophoblast invasion

Nitric oxide regulates many important cellular processes including motility and invasion. Many of its effects are mediated through the modification of specific cysteine residues in target proteins, a process called S-nitrosylation. Here we show that S-nitrosylation of proteins occurs at the leading edge of migrating trophoblasts and can be attributed to the specific enrichment of inducible nitric oxide synthase (iNOS/NOS2) in this region. Localisation of iNOS to the leading edge is co-incidental with a site of extensive actin polymerisation and is only observed in actively migrating cells. In contrast endothelial nitric oxide synthase (eNOS/NOS3) shows distribution that is distinct and non-colocalised with iNOS, suggesting that the protein S-nitrosylation observed at the leading edge is caused only by iNOS and not eNOS. We have identified MMP-9 as a potential target for S-nitrosylation in these cells and demonstrate that it co-localises with iNOS at the leading edge of migrating cells. We further demonstrate that iNOS plays an important role in promoting trophoblast invasion, which is an essential process in the establishment of a successful pregnancy.

Extracellular disulfide exchange and the regulation of cellular function

An emerging concept is that disulfide bonds can act as a dynamic scaffold to present mature proteins in different conformational and functional states on the cell surface. Two examples are the conversion of the receptor, integrin a alpha(IIb)beta(3), from a low affinity to a high affinity state, and the interaction of CD4 receptor with the HIV-1 envelope glycoprotein gp120 to promote virus-cell fusion. In both of these cases there is a remodeling of the protein disulfide bonding pattern. The formation and rearrangement of disulfide bonds is modulated by a family of enzymes known as the thiol isomerases, which include protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72. While these enzymes were reported originally to be restricted in location to the endoplasmic reticulum, in some cells thiol isomerases are found on the cell surface. This may indicate a wider role for these enzymes in cell function. In platelets it has been shown that reagents that react with cell surface sulfhydryl groups are capable of blocking a number of functional responses, including integrin-mediated aggregation, adhesion, and granule secretion. Furthermore, the use of function blocking antibodies to either PDI or ERp5 causes inhibition of these functional responses. This review summarizes current knowledge of the extracellular regulation of disulfide exchange and the implications of this in the regulation of cell function.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

The structure of echovirus type 12 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD 55)

Echovirus type 12 (EV12), an enterovirus of the Picornaviridae family, uses the complement regulator, decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF, consisting of short consensus repeat domains 3 and 4, from cryo-negative stain electron microscopy data (EMD #1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the two-fold symmetry axes. Despite this general similarity, our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF34 and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB #1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF-binding phenotype in these viruses.

Wormlike micelle formation and flow alignment of a pluronic block copolymer in aqueous solution

The self-assembly into wormlike micelles of a poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) triblock copolymer Pluronic P84 in aqueous salt solution (2 M NaCl) has been studied by rheology, small-angle X-ray and neutron scattering (SAXS/SANS), and light scattering. Measurements of the flow curves by controlled stress rheometry indicated phase separation under flow. SAXS on solutions subjected to capillary flow showed alignment of micelles at intermediate shear rates, although loss of alignment was observed for high shear rates. For dilute solutions, SAXS and static light scattering data on unaligned samples could be superposed over three decades in scattering vector, providing unique information on the wormlike micelle structure over several length scales. SANS data provided information on even shorter length scales, in particular, concerning "blob" scattering from the micelle corona. The data could be modeled based on a system of semiflexible self-avoiding cylinders with a circular cross-section, as described by the wormlike chain model with excluded volume interactions. The micelle structure was compared at two temperatures close to the cloud point (47 degrees C). The micellar radius was found not to vary with temperature in this region, although the contour length increased with increasing temperature, whereas the Kuhn length decreased. These variations result in an increase of the low-concentration radius of gyration with increasing temperature. This was consistent with dynamic light scattering results, and, applying theoretical results from the literature, this is in agreement with an increase in endcap energy due to changes in hydration of the poly(ethylene oxide) blocks as the temperature is increased.

A SAXS study of flow alignment of thermotropic liquid crystal mixtures

We report on the capillary flow behaviour of thermotropic liquid crystal mixtures containing 4-n-octyl-4'-cyanobiphenyl (8CB) and 4-n-pentyl-4'-cyanobiphenyl (5CB). The liquid crystal mixtures are studied in the Nematic (N) and Smectic (SA) phases at room temperature. Polarised optical microscopy (POM), rheology and simultaneous X-ray diffraction (XRD)/capillary flow experiments are performed to characterise the system. Polarised optical microscopy reveals a dramatic change in optical texture when the 5CB content is increased from 20 to 30% in the mixtures. X-ray diffraction results show that the system goes through a SA-N phase transition, such that the mixtures are smectic for 10-20% 5CB and nematic for 30-90% 5CB. Smectic mixtures flow with the layers aligned along the flow direction (mesogens perpendicular to flow) while nematic mixtures flow with the mesogens aligned in the flow direction. Simultaneous XRD/shear flow experiments show that the SA-N transition is independent of the flow rate in the range 1-6 ml min-1. The correlation length of the liquid crystal order decreases with increasing 5CB content. Rheology is used to prove that the correlation length behaviour is related to a reduction in the viscosity of the mixture.