The Carboxyl-Terminal Segment of Apolipoprotein A-V Undergoes a Lipid-Induced Conformational Change

Apolipoprotein (apo) A-V is a 343-residue, multidomain protein that plays an important role in regulation of plasma triglyceride homeostasis. Primary sequence analysis revealed a unique tetraproline sequence (Pro293-Pro296) near the carboxyl terminus of the protein. A peptide corresponding to the 48-residue segment beyond the tetraproline motif was generated from a recombinant apoA-V precursor wherein Pro295 was replaced by Met. Cyanogen bromide cleavage of the precursor protein, followed by negative affinity chromatography, yielded a purified peptide. Nondenaturing polyacrylamide gel electrophoresis verified that apoA-V(296-343) solubilizes phospholipid vesicles, forming a relatively heterogeneous population of reconstituted high-density lipoprotein with Stokes’ diameters\u3e17 nm. At the same time, apoA-V(296-343) failed to bind a spherical lipoprotein substrate in vitro. Far-UV circular dichroism spectroscopy revealed the peptide is unstructured in buffer yet adopts significant R-helical secondary structure in the presence of the lipid mimetic solvent trifluoroethanol (TFE; 50% v/v). Heteronuclear multidemensional NMR spectroscopy experiments were conducted with uniformly 15N- and 15N/13C-labeled peptide in 50% TFE. Peptide backbone assignment and secondary structure prediction using TALOSþ reveal the peptide adopts R-helix secondary structure from residues 309 to 334. In TFE, apoA-V(296-343) adopts an extended amphipathic R-helix, consistent with a role in lipoprotein binding as a component of full-length apoA-V

Simulation and Measurement of Transient Fluid Phenomena within Diesel Injection

Rail pressures of modern diesel fuel injection systems have increased significantly over recent years, greatly improving atomisation of the main fuel injection event and air utilisation of the combustion process. Continued improvement in controlling the process of introducing fuel into the cylinder has led to focussing on fluid phenomena related to transient response. High-speed microscopy has been employed to visualise the detailed fluid dynamics around the near nozzle region of an automotive diesel fuel injector, during the opening, closing and post injection events. Complementary computational fluid dynamic (CFD) simulations have been undertaken to elucidate the interaction of the liquid and gas phases during these highly transient events, including an assessment of close-coupled injections. Microscopic imaging shows the development of a plug flow in the initial stages of injection, with rapid transition into a primary breakup regime, transitioning to a finely atomised spray and subsequent vaporisation of the fuel. During closuring of the injector the spray collapses, with evidence of swirling breakup structures together with unstable ligaments of fuel breaking into large slow-moving droplets. This leads to sub-optimal combustion in the developing flame fronts established by the earlier, more fully-developed spray. The simulation results predict these observed phenomena, including injector surface wetting as a result of large slow-moving droplets and post-injection discharge of liquid fuel. This work suggests that post-injection discharges of fuel play a part in the mechanism of the initial formation, and subsequent accumulation of deposits on the exterior surface of the injector. For multiple injections, opening events are influenced by the dynamics of the previous injection closure; these phenomena have been investigated within the simulations

Contrast in chloride exclusion between two grapevine genotypes and its variation in their hybrid progeny

Potted grapevines of 140 Ruggeri (Vitis berlandieri × Vitis rupestris), a good Cl− excluder, and K 51-40 (Vitis champinii × Vitis riparia ‘Gloire’), a poor Cl− excluder, and of a family obtained by crossing the two genotypes, were used to examine the inheritance of Cl− exclusion. Rooted leaves were then used to further investigate the mechanism for Cl− exclusion in 140 Ruggeri. In both a potting mix trial (plants watered with 50 mM Cl−) and a solution culture trial (plants grown in 25 mM Cl−), the variation in Cl− accumulation was continuous, indicating multiple rather than single gene control for Cl− exclusion between hybrids within the family. Upper limits of 42% and 35% of the phenotypic variation in Cl− concentration could be attributed to heritable sources in the potting mix and solution culture trials, respectively. Chloride transport in roots of rooted leaves of both genotypes appeared to be via the symplastic pathway, since addition of 8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS), an apoplastic tracer, revealed no obvious PTS fluorescence in the laminae of either genotype, despite significant accumulation of Cl− in laminae of K 51-40 during the PTS uptake period. There was no significant difference in either unidirectional 36Cl− flux (10 min) or 36Cl− uptake (3 h) into roots of rooted leaves exposed to 5, 10, or 25 mM Cl−. However, the percentage of 36Cl− transported to the lamina (3 h) was significantly lower in 140 Ruggeri than in K 51-40, supporting reduced Cl− loading into xylem and implicating the root stele in the Cl− exclusion mechanism

Variation between and within grapevine families in reaction to leaf inoculation with downy mildew sporangia under controlled conditions

Vine reaction to downy mildew [Plasmopara viticola (Berk. & Curt.) Berl. & de Toni] inoculation was investigated between and within seven full-sib grapevine families under controlled culture conditions. Families were generated by crossing resistant x susceptible and susceptible x susceptible genotypes. Leaf infection following downy mildew inoculation was assessed using cultured leafed single node cuttings under controlled conditions. The severity of disease infection 7 days after inoculation was based on the expression of leaf chlorosis and sporulation symptoms using a 1 to 9 scale where a score of 1 meant there was no visible sign of infection and 9 meant > 80 % of the leaf area was infected. A measure of the hypersensitive response (discrete necrotic spots) was also used to rank vine reaction to inoculation. Hybrids within families varied widely in their reaction to inoculation. Any hybrid that received a mean symptom expression score ≤ 3, which was equivalent to or less than that assigned to 'Chambourcin', was classified as downy mildew resistant. The proportion of vines within families scored as resistant ranged from 4.6 to 22.5 % and from 4.6 to 47.4 % for leaf chlorosis and sporulation, respectively, between crosses. There was a strong correlation between leaf chlorosis and sporulation expression within each family (r2 ranged from 0.6 to 0.8). The number of resistant hybrids selected within families by the combined symptoms of leaf chlorosis and sporulation ranged from 3 to 7. Depending on family, segregation of resistant to susceptible phenotypes based on symptoms of leaf chlorosis and sporulation fitted 1:15, 1:7, 1:3 or 1:1 ratios. Segregation for hypersensitive reaction (HR) to non-HR fitted 1:1, 1:2 or 1:3 ratios within families. Hybrids that displayed the HR had mean scores for leaf chlorosis and sporulation less than those not displaying the necrotic spots characteristic of the reaction in four of the families investigated. The results are discussed in terms of the inheritance of resistance and the development of a strategy for future breeding.

Vitis seed longevity after prolonged cold storage

Research Not

A method using leafed single-node cuttings to evaluate downy mildew resistance in grapevine

A method using leafed single-node cuttings, incubated at 25 ± 2 °C and 100 % humidity, was examined to screen grapevine genotypes for resistance to downy mildew (Plasmopara viticola (Bert. & Curt.) Berl. & de Toni). Cuttings were taken at the 4th, 5th and 6th nodes back from apices of actively growing shoots. Disease symptoms, namely chlorosis, sporulation and necrosis, were observed on the leaves of cuttings within 6 days of incubation after inoculation. Based on chlorosis and sporulation, genotypes were ranked from highly susceptible to resistant in the order of Sultana, M46-32 (Bicane x Villard blanc), Joannes Seyve 23.416, Marroo Seedless and Chambourcin. The expressions of both symptoms increased with the concentration of inocula applied to leaves (1 x 105 and 5 x 104 sporangia per ml), but the overall genotypic ranking was unaltered. The third symptom of leaf necrosis occurred on infected leaves either as progressively enlarging dead areas or as smaller localised necrotic spots. The latter has been call ed the ‘necrotic response’ symptom and its expression depended on genotype, although its value for separating or ranking genotypes was unreliable. Leaf chlorosis was associated with leaf sporulation (r2=0.41-0.47). Genotypes with necrotic response exhibited reduced leaf chlorosis and sporulation. A comparison between this new leafed singlenode cutting method and a previously published leaf disc method indicated it was more reliable for separating genotypes for downy mildew resistance.

Critical holes in undercooled wetting layers

The profile of a critical hole in an undercooled wetting layer is determined by the saddle-point equation of a standard interface Hamiltonian supported by convenient boundary conditions. It is shown that this saddle-point equation can be mapped onto an autonomous dynamical system in a three-dimensional phase space. The corresponding flux has a polynomial form and in general displays four fixed points, each with different stability properties. On the basis of this picture we derive the thermodynamic behaviour of critical holes in three different nucleation regimes of the phase diagram.Comment: 18 pages, LaTeX, 6 figures Postscript, submitted to J. Phys.

Nitrogen transactions along the digestive tract of lambs concurrently infected with Trichostrongylus colubriformis and Ostertagia circumcincta

Twelve lambs, paired on the basis of live weight, were cannulated in the abomasum, in the proximal jejunum approximately 4 m distal to the pylorus and in the terminal ileum. Six were infected with 3000 Trichostrongylus colubriformis and 3000 Ostertagia circumcincta larvae each day for 18 weeks and the remainder were pair-fed to individual infected lambs. All animals were offered ryegrass (Lolium perenne)–white clover (Trifolium repens) pasture, cut daily. Dry matter (DM) intake, live weight, faecal egg concentration, plasma pepsinogen and plasma protein concentrations were measured weekly. During weeks 7 and 17 after commencement of infection, the flow of digesta along the gastrointestinal tract was measured together with enteric plasma loss and true digestion and absorption of ¹²⁵I-labelled albumin in the small intestine. DM intake was depressed by parasitism, being 1331, (se 70), 423 (se 32) and 529 (se 52) g/d during weeks 3, 7 and 17 respectively. The flow of nitrogen at the proximal jejunum and in faeces was increased by parasitism during week 7 and at the abomasum and ileum during week 17. Plasma protein-N loss (g/d) into the gastrointestinal tract was 0.68 (se 0.091) and 1.97 (se 0.139) during week 7, and 0.85 (se0.158) and 1.96 (se 0.396) during week 17, in control and infected sheep respectively. True digestion and absorption of albumin in the proximal small intestine, the site of infection, was very low (mean 0.08) and was not affected by parasitism. Between the abomasum and terminal ileum absorption of albumin was high (mean 0.87) and again was not affected by parasitism. It was calculated that of the total increase in endogenous protein passing from the ileum tract as a result of infection, plasma protein comprised only a small percentage (10–36%). The major proportion of digestion and absorption of protein occurred in the distal small intestine beyond the site of infection and was not affected by infection

Site percolation and random walks on d-dimensional Kagome lattices

The site percolation problem is studied on d-dimensional generalisations of the Kagome' lattice. These lattices are isotropic and have the same coordination number q as the hyper-cubic lattices in d dimensions, namely q=2d. The site percolation thresholds are calculated numerically for d= 3, 4, 5, and 6. The scaling of these thresholds as a function of dimension d, or alternatively q, is different than for hypercubic lattices: p_c ~ 2/q instead of p_c ~ 1/(q-1). The latter is the Bethe approximation, which is usually assumed to hold for all lattices in high dimensions. A series expansion is calculated, in order to understand the different behaviour of the Kagome' lattice. The return probability of a random walker on these lattices is also shown to scale as 2/q. For bond percolation on d-dimensional diamond lattices these results imply p_c ~ 1/(q-1).Comment: 11 pages, LaTeX, 8 figures (EPS format), submitted to J. Phys.