The influence of different lipid environments on the structure and function of the hepatitis C virus p7 ion channel protein

Abstract

The hepatitis C virus (HCV) encodes the p7 protein that oligomerizes to form an ion channel. The 63 amino acid long p7 monomer is an integral membrane protein predominantly found in the endoplasmic reticulum (ER). Although it is currently unknown whether p7 is incorporated into secreted virions, its presence is crucial for the release of infectious virus. The molecular and biophysical mechanism employed by the p7 ion channel is largely unknown, but in vivo it is likely to be embedded in membranes undergoing changes in lipid composition. In this study we analyze the influence of the lipid environment on p7 ion channel structure and function using electrophysiology and synchrotron radiation circular dichroism (SRCD) spectroscopy. We incorporated chemically synthesized p7 polypeptides into artificial planar membranes of various lipid compositions. A lipid bilayer composition comprising phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (4:1 PC:PE) led to burst-like patterns in the channel recordings with channel openings lasting up to 0.5 s. The reverse ratio of PC:PE (1:4) gave rise to individual channels continuously opening for up to 8 s. SRCD spectroscopy of p7 embedded into liposomes of corresponding lipid compositions suggests there is a structural effect of the lipid composition on the p7 protein.     

A Molecular Dynamics Investigation of Chain Conformational Changes In Compressed Bilayers

Abstract

The conformations of the chains constituting the hydrophilic component of alkyl monolayers and bilayers are investigated by performing molecular dynamics atomistic simulations on these systems at different temperatures. Several monitoring techniques are used to reveal the chain conformations, including atom pair radial distribution functions, evolutions of the torsional angles over thousands of timesteps, frequency distributions of the torsionl angles and ‘snapshot’ plots of the atomic configurations. These methods consistently testify to the stability of the trans (fully extended) character of the strain-free alkyl chains up to room temperature. The chains retain much of this conformation even when the layers are compressed by the application of pressure, to which the chains respond by ‘folding’ at the ends attaching them to the substrate planes while maintaining directions which are mainly normal to these planes. A non-zero gap between the layers is also maintained. A pressure of about 50 kbar abruptly causes all motion in the chains to cease, resulting in a highly ordered lattice structure.     

Fish community response to the longitudinal environmental gradient in Czech deep-valley reservoirs: Implications for ecological monitoring and management

Ecological quality assessment of non-natural water bodies is, in contrast to natural systems, less developed and requires determining biological indicators that reliably reflect environmental conditions and anthropogenic pressures. This study was motivated to propose fish indicators appropriate for assessment of reservoir ecosystems in central Europe. We analysed changes in water quality, total biomass and the taxonomic, trophic and size composition of fish communities along the longitudinal axes of four elongated, deep-valley reservoirs. Due to high nutrient inputs from their catchments, the reservoirs exhibited pronounced within-system gradients in primary productivity and water transparency. Although fish communities were similar among the reservoirs and dominated by few native species, the community structure and biomass systematically changed along the longitudinal axes of the reservoirs. The biomass and proportion of planktivores/benthivores in the fish community were highest at eutrophic sites near the river inflow and declined substantially towards deep, more oligotrophic sites close to the dam. The biomass and proportion of piscivores significantly increased downstream within the reservoirs alongside improving water quality. At species level, perch Perca fluviatilis and bream Abramis brama responded most sensitively, although in opposite directions, to the longitudinal environmental gradient. The major longitudinal changes in fish community characteristics were found to be consistent between pelagic and benthic habitats. The results of this study suggest that fish communities are appropriate indicators of eutrophication and can be used for ecological quality assessment of non-natural lentic water bodies, such as reservoirs. Moreover, our results underline the necessity to consider within-system gradients in water quality and the fish community when planning sampling programmes for deep-valley reservoirs.     

EXPERIMENTAL EVOLUTION OF RESISTANCE IN BRASSICA RAPA: CORRELATED RESPONSE OF TOLERANCE IN LINES SELECTED FOR GLUCOSINOLATE CONTENT

The evolutionary response of plant populations to selection for increased defense may be constrained by costs of defense. The purpose of this study was to investigate such constraints on the evolution of defense due to a cost of defense manifested as a trade-off between defense and tolerance. Variation in the response to artificial damage (tolerance) among lines of Brassica rapa that had been artificially selected for foliar glucosinolate content (defense) was examined. Leaf area was removed from replicates of three selection lines (high glucosinolates, control, and low glucosinolates) at three damage levels (0%, 20%, and 60% damage). An external cost of defense would result in a statistically significant selection line by damage treatment interaction, with those selected for high defense expressing less tolerance than those selected for low defense. Damage treatment had a significant overall effect on estimated total fitness, with fitness declining with increasing damage level. Further, selection line also had a significant overall effect on estimated total fitness, with low-defense selection lines having higher fitness compared to both control and high-defense selection lines. More importantly, a cost of defense in terms of tolerance was demonstrated by a significant selection line-by-damage treatment interaction. This interaction was in the direction to demonstrate a genetic trade-off between defense and tolerance, with low-defense selection lines decreasing estimated total fitness in response to damage less than both control and high-defense selection lines. Variation in tolerance among selection lines was due to the greater ability of low-defense lines to maintain fruit and seed production despite the presence of damage. In terms of tolerance, this cost of glucosinolate production in B. rapa could constrain the evolution of increased defense and, in so doing, maintain individuals within the population that are poorly defended yet tolerant.     

Gramicidin A-phospholipid model systems

Gramicidin A forms ion-conducting channels which can traverse the hydrocarbon core of lipid bilayer membranes. The structures formed by gramicidin A are among the best characterized of all membrane-bound polypeptides or proteins. In this review a brief summary is given of the occurrence, conformation, and synthesis of gramicidin A, and of its use as a model for ion transport and the interaction of proteins and lipids in biological membranes.     

Bounding fluid viscosity and translational diffusion in a fluid lipid bilayer

Fluorescence recovery after photobleaching was used to investigate the translational diffusion of a fluorescent derivative of a membrane-spanning lipid in L phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine prepared in water and in glycerol. The translational diffusion coefficient in hydrated bilayers (D _w) ranged between 2 and 5x10^–8 cm²/s and in glycerinated bilayers (D _g) the range was between 3 and 24×10^–10 cm²/s between 10° and 40°C. These results are discussed in terms of models for diffusion in membranes.     

Gating processes of channels induced by colicin A,its C-terminal fragment and colicin E1 in planar lipid bilayers

The dependence on pH and membrane potential of the pore formed by colicin A and its C-terminal 20 kDa fragment has been measured using planar lipid bilayers. The single channel conductance of the pore formed by both colicin A and the fragment increases with pH with an apparent pK of 6.0. At pH 5.0 the gating by membrane potential of the channels formed by either colicin A or its fragment is identical. At the same pH, quite similar pore properties were found when using the related bacteriocin, colicin E₁. In agreement with previous studies, these data indicate that the protein structure containing the lumen of the pore resides in the 20 kDa C-terminal part of the colicin A and favours the recently proposed model, based on protein sequence analysis, which proposes that colicin A, E₁ and I_B C-terminal domains are folded in the same three-dimensional structure. However, it is also shown that colicin A and not its C-terminal fragment undergoes a pH dependent transition between an acidic and a basic form of the pore with an apparent pK of 5.3. The two forms of the pore differ by their gating charge but not by the channel size. These results suggest that there is a pH dependent association between the C-terminal domain carrying the lumen of the pore and another domain of the molecule which affect the pore sensitivity to membrane potential.     

Delayed inducible resistance against a leaf-chewing insect in four deciduous tree species

Summary Both mechanical damage to mountain birch foliage and rearing of moth larvae on the trees reduced the growth of Epirrita autumnata larvae reared on these trees in the following year. The effects of physical damage and some other cues from insects were additive. On bird cherry the performance of Epirrita larvae was equal on untreated trees and on trees artificially defoliated in the previous year, but larval growth was reduced on previously insect-damaged branches. With mountain ash just physical damage per se reduced the performance of Epirrita larvae. On Salix phylicifolia there were no significant differences in the growth or survival of Epirrita on untreated control bushes and on bushes with partial larval damage during the previous year. Among untreated control trees the growth and survivorship of Epirrita were higher on fast-growing willow and bird cherry than on the slow-growing mountain birch. Mountain birch and mountain ash, the two deciduous tree species adapted to nutrient-poor soils, showed delayed inducible resistance triggered by defoliation (artificial or insect-made). This supports the hypothesis that delayed inducible resistance may be a passive response due to nutrient-stress caused by defoliation. On the other hand, the additional increase in the resistance of mountain birch triggered by specific cues from insects suggests that this response may be an evolved defense against leaf-eating insects.     

Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM Black lipid membranes - CCCP carbonyl cyanide m-chlorophenylhydrazone - DOPC dioleoyl phosphatidylcholine - PS phosphatidylserine - TPP⁺ tetraphenylphosphonium cation     

Artificial seeds in barley: encapsulation of microspore-derived embryos

Summary An in vitro culture system has been developed for barley (Hordeum vulgare), which yields high frequencies of high quality microspore-derived embryos without an intervening callus phase. The embryos are very similar to zygotic embryos with regard to their morphology and germination capacity. These embryos were encapsulated in sodium alginate to produce individual beads containing one embryo each. In accordance with the literature, these beads are denoted artificial seeds. The artificial seeds germinated well and with a root system superior to that of non-encapsulated embryos. The artificial seeds also maintained their germination capacity for at least 6 months, whereas non-encapsulated embryos did not survive more than 2 weeks in storage. Artificial seeds, thus, probably provide a simple and universal delivery system of in vitro plantlets to greenhouse or field.