Identification of a discrete intermediate in the assembly disassembly of physalis mottle tymovirus through mutational analysis

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T = 3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T = 3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T = 3 capsid assembly, in contrast, the deletion of even a single residue from the C terminus (PhN188 Delta 1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75A and D144 are not crucial for the T = 3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188 Delta 1, pR PhH69A and pR PhK143E mutant proteins.

Structures of three different neutral polysaccharides of Acinetobacter baumannii, NIPH190, NIPH201, and NIPH615, assigned to K30, K45, and K48 capsule types, respectively, based on capsule biosynthesis gene clusters

Neutral capsular polysaccharides (CPSs) were isolated from Acinetobacter baumannii NIPH190, NIPH201, and NIPH615. The CPSs were found to contain common monosaccharides only and to be branched with a side-chain 1→3-linked β-d-glucopyranose residue. Structures of the oligosaccharide repeat units (K units) of the CPSs were elucidated by 1D and 2D 1H and 13C NMR spectroscopy. Novel CPS biosynthesis gene clusters, designated KL30, KL45, and KL48, were found at the K locus in the genome sequences of NIPH190, NIPH201, and NIPH615, respectively. The genetic content of each gene cluster correlated with the structure of the CPS unit established, and therefore, the capsular types of the strains studied were designated as K30, K45, and K48, respectively. The initiating sugar of each K unit was predicted, and glycosyltransferases encoded by each gene cluster were assigned to the formation of the linkages between sugars in the corresponding K unit.

Theoretical-Studies on the Modes of Binding of Some of the Beta-Glycosidically Linked Glucobioses to Concanavalin-A

The probable modes of binding for methyl-α-d-sophoroside, methyl-β-d-sophoroside, laminariboise and cellobiose to concanavalin A have been determined using theoretical methods. Methyl-d-sophorosides can bind to concanavalin A in two modes, i.e. by placing their reducing as well as non-reducing sugar units in the carbohydrate specific binding site, whereas laminaribiose and cellobiose can reach the binding site only with their non-reducing glucose units. However, the probability for methyl-α-d-sophoroside to bind to concanavalin A with its reducing sugar residue as the occupant of the binding site is much higher than it is with its non-reducing sugar residue as the occupant of the sugar binding site. A few of the probable conformers of methyl-β-d-sophoroside can bind to concanavalin A with either the reducing or non-reducing glucose unit. Higher energy conformers of cellobiose or laminaribiose can reach the binding site with their non-reducing residues alone. The relative differences in the binding affinities of these disaccharides are mainly due to the differences in the availability of proper conformers which can reach the binding site and to non-covalent interactions between the sugar and the protein. This study also suggests that though the sugar binding site of concanavalin A accommodates a single sugar residue, the residue outwards from the binding site also interacts with concanavalin A, indicating the existence of extended concanavalin A carbohydrate interactions.

Gibbs Energy of Formation of Cobalt Divanadium Tetroxide

The Gibbs energy of formation of V2O3-saturated spinel CoV2O4 has been measured in the temperature range 900–1700 K using a solid state galvanic cell, which can be represented as Pt, Co + CoV2O4 + V2O3/(CaO) ZrO2/Co + CoO, Pt. The standard free energy of formation of cobalt vanadite from component oxides can be represented as CoO (rs) + V2O3 (cor) → CoV2O4 (sp), ΔG° = −30,125 − 5.06T (± 150) J mole−1. Cation mixing on crystallographically nonequivalent sites of the spinel is responsible for the decrease in free energy with increasing temperature. A correlation between “second law” entropies of formation of cubic 2–3 spinels from component oxides with rock salt and corundum structures and cation distribution is presented. Based on the information obtained in this study and trends in the stability of aluminate and chromite spinels, it can be deduced that copper vanadite is unstable.

The crystal structure of the amino-terminal pentapeptide of suzukacillin. Occurrence of a four-fold peptide helix

The monohydrate of the protected amino-terminal pentapeptide of suzukacillin, t-butoxycarbonyl--aminoisobutyryl-L-prolyl-L-valyl--aminoisobutyryl-L-valine methyl ester, C29H51N5O8, crystallizes in the orthorhombic space group P212121 with a= 10.192, b= 10.440, c= 32.959 Å, and Z= 4. The structure has been solved by direct methods and refined to an R value of 0.101 for 1 827 observed reflections. The molecule exists as a four-fold helix with a pitch of 5.58 Å. The helix is stabilised by N–H O hydrogen bonds, two of the 51 type (corresponding to the -helix) and the third of the 41 type (310 helix). The carbonyl oxygen of the amino-protecting group accepts two hydrogen bonds, one each from the amide NH groups of the third (41) and fourth (51) residues. The remaining 51 hydrogen bond is between the two terminal residues. The lone water molecule in the structure is hydrogen bonded to carbonyl oxygens of the prolyl residue in one molecule and the non-terminal valyl residue in a symmetry-related molecule.

Alternatives to sodium metabisulphite for blackspot prevention in prawns

In Australia, prawns are usually treated with a 1% sodium metabisulphite solution to prevent black spot. Two alternatives, Bacterol and Snow Fresh, were compared to the standard metabisulphite treatment used by industry. Bacterol gave similar protection to sodium metabisulphite, while Snow Fresh showed potential as a substitute. The concentrations most appropriate were determined from residue levels after treatment.

Pesticide residues in food - acute dietary exposure

Consumer risk assessment is a crucial step in the regulatory approval of pesticide use on food crops. Recently, an additional hurdle has been added to the formal consumer risk assessment process with the introduction of short-term intake or exposure assessment and a comparable short-term toxicity reference, the acute reference dose. Exposure to residues during one meal or over one day is important for short-term or acute intake. Exposure in the short term can be substantially higher than average because the consumption of a food on a single occasion can be very large compared with typical long-term or mean consumption and the food may have a much larger residue than average. Furthermore, the residue level in a single unit of a fruit or vegetable may be higher by a factor (defined as the variability factor, which we have shown to be typically ×3 for the 97.5th percentile unit) than the average residue in the lot. Available marketplace data and supervised residue trial data are examined in an investigation of the variability of residues in units of fruit and vegetables. A method is described for estimating the 97.5th percentile value from sets of unit residue data. Variability appears to be generally independent of the pesticide, the crop, crop unit size and the residue level. The deposition of pesticide on the individual unit during application is probably the most significant factor. The diets used in the calculations ideally come from individual and household surveys with enough consumers of each specific food to determine large portion sizes. The diets should distinguish the different forms of a food consumed, eg canned, frozen or fresh, because the residue levels associated with the different forms may be quite different. Dietary intakes may be calculated by a deterministic method or a probabilistic method. In the deterministic method the intake is estimated with the assumptions of large portion consumption of a ‘high residue’ food (high residue in the sense that the pesticide was used at the highest recommended label rate, the crop was harvested at the smallest interval after treatment and the residue in the edible portion was the highest found in any of the supervised trials in line with these use conditions). The deterministic calculation also includes a variability factor for those foods consumed as units (eg apples, carrots) to allow for the elevated residue in some single units which may not be seen in composited samples. In the probabilistic method the distribution of dietary consumption and the distribution of possible residues are combined in repeated probabilistic calculations to yield a distribution of possible residue intakes. Additional information such as percentage commodity treated and combination of residues from multiple commodities may be incorporated into probabilistic calculations. The IUPAC Advisory Committee on Crop Protection Chemistry has made 11 recommendations relating to acute dietary exposure.

Electroleaching of Zinc leach residues

A room-temperature cathodic electrolytic process was developed in the laboratory to recover zinc from industrial leach residues. The various parameters affecting the electroleaching process were studied using a statistically designed experiment. To understand the mechanisms behind the electrode processes, cyclic voltammetry and galvanostatic studies were carried out. The role of Einh measurements in monitoring such an electroleaching procedure is also shown. Since significant amounts of iron were also present in the leach liquor, attempts were made to purify it before zinc recovery by electrowinning. Reductive dissolution and creation of anion vacancies were found to be responsible for the dissolution of zinc ferrite present in the leach residue. A flow sheet of the process is given.

Management of white grubs (Coleoptera: Scarabaeidae) on groundnut in southern India

The chemical control of groundnut white grubs, Holotrichia serrata F. and H. reynaudi Blanchard (Coleoptera: Scarabaeidae), was studied in south--central India. Microplot trials demonstrated that chlorpyrifos and imidacloprid seed--dressings were effective against H. serrata at rates as low as 0.6 and 3.5 g a.i. kg-1, respectively, while microplot and on--farm trials showed that 1.2 and 3.5 g a.i. kg-1of chlorpyrifos and imidacloprid, respectively, were required for H. reynaudi. Chlorpyrifos residue analyses indicated that at 20 days after sowing (d.a.s.) rates up to 5.0 g a.i. kg-1 produced residues in soil and groundnut seedlings markedly below the relevant MRL, and no detectable residues at harvest under the southern Indian rainy--season environment. A farmer survey found that in Andhra Pradesh (AP), insecticides (chlorpyrifos and phorate) were applied for white grub control in 37.5% of farms sampled, while no insecticides were applied for this purpose in Karnataka and Tamil Nadu. The white grub density on farms in AP where insecticide had been applied averaged 0.07 larvae m-2, compared to 1.04 larvae m-2 in the remaining AP farms. In AP, Karnataka and Tamil Nadu, 70%, 42% and 39% of currently untreated groundnut fields, respectively, exceed the provisional economic threshold. A survey in the Anantapur district of AP found that farmer’s target and achieved rates for seed treatment averaged 0.44 and 0.52 g a.i. kg-1, both below optimal rates determined in microplot experiments. These data provide the foundation for an effective and sustainable program of management for groundnut white grubs in south--central India by providing key efficacy data and baseline data on farmer insecticide- use patterns.

Alamethicin and related membrane channel forming polypeptides

Alamethicin and several related microbial polypeptides, which contain a high proportion of agr-aminoisobutyric acid (Aib) residues, possess the ability to modify the permeability properties of phospholipid bilayer membranes. Alamethicin induces excitability phenomena in model membranes and has served as an excellent model for the study of voltage sensitive transmembrane channels. This review summarizes various aspects of the structural chemistry and membrane modifying properties of alamethicin and related Alb containing peptides. The presence of Aib residues in these sequences, constrains the polypeptides to 310 or agr-helical conformations. Functional membrane channels are formed by aggregation of cylindrical peptide helices, which span the lipid bilayer, forming a scaffolding for an aqueous column across the membrane. After consideration of the available data on the conductance characteristics of alamethicin channels, a working, hypothesis for a channel model is outlined. Channel aggregates in the lipid phase may be stabilized by intermolecular hydrogen bonding, involving a central glutamine residue and also by interactions between the macro-dipoles of proximate peptide helices. Fluctuations between different conductance states are rationalized by transitions between states of different aggregation and hence altered dimensions of the aqueous core or by changes in net dipole moment of the aggregate. Ion fluxes through the channel may also be affected by the electric field within the aqueous core.

Conformations Of Peptides Containing 1-Aminocyclohexane-Carboxylic Acid (Acc6) - Crystal-Structures Of 2 Model Peptides

The crystal structures of two peptides containing 1-aminocyclohexanecarboxylic acid (Acc6) are described. Boc-Aib-Acc6-NHMe · H2O adopts a β-turn conformation in the solid state, stabilized by an intramolecular 4 → 1 hydrogen bond between the Boc CO and methylamide NH groups. The backbone conformational angles (φAib = – 50.3°, ψAib = – 45.8°; φAcc6 = – 68.4°, ψAcc6 = – 15°) lie in between the values expected for ideal Type I or III β-turns. In Boc-Aib-Acc6-OMe, the Aib residue adopts a partially extended conformation (φAib = – 62.2°, ψAib = 143°) while the Acc6residue maintains a helical conformation (φAcc6 = 48°, ψAcc6= 42.6°). 1H n.m.r. studies in CDCl3 and (CD3)2SO suggest that Boc-Aib-Acc6-NHMe maintains the β-turn conformation in solution.

Long-term persistence and efficacy of spinosad against Rhyzopertha dominica (Coleoptera : Bostrychidae) in wheat

A laboratory study was undertaken to determine the persistence and efficacy of spinosad against Rhyzopertha dominica (F.) in wheat stored for 9 months at 30 degrees C and 55 and 70% relative humidity. The aim was to investigate the potential of spinosad for protecting wheat from R. dominica during long-term storage in warm climates. Wheat was treated with spinosad at 0.1, 0.5 and 1 mg kg(-1) grain and sampled after 0, 1.5, 3, 4.5, 6, 7.5 and 9 months of storage for bioassays and residue analyses. Residues were estimated to have declined by 30% during 9 months of storage at 30 degrees C and there was no effect of relative humidity. Spinosad applied at 0.5 or 1 mg kg(-1) was completely effective for 9 months, with 100% adult mortality after 14 days of exposure and no five F, adults produced. Adult mortality was < 100% in some samples of wheat treated with 0.1 mg kg(-1) of spinosad, and live progeny were produced in all samples treated at this level. The results show that spinosad is likely to be an effective grain protectant against R. dominica in wheat stored in warm climates.

Pasteurella multocida Expresses Two Lipopolysaccharide Glycoforms Simultaneously, but Only a Single Form Is Required for Virulence: Identification of Two Acceptor-Specific Heptosyl I Transferases

Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-D-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo1 residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures.

Conserved water-mediated H-bonding dynamics of catalytic Asn 175 in plant thiol protease

The role of invariant water molecules in the activity of plant cysteine protease is ubiquitous in nature. On analysing the 11 different Protein DataBank (PDB) structures of plant thiol proteases, the two invariant water molecules W I and W2 (W220 and W222 in the template 1PPN structure) were observed to form H-bonds with the Ob atom of Asn 175. Extensive energy minimization and molecular dynamics simulation studies up to 2 ns on all the PDB and solvated structures clearly revealed the involvement of the H-bonding association of the two water molecules in fixing the orientation of the asparagine residue of the catalytic triad. From this study, it is suggested that H-bonding of the water molecule at the W1 invariant site better stabilizes the Asn residue at the active site of the catalytic triad.

Amelioration of soil compaction can take 5 years on a Vertisol under no till in the semi-arid subtropics

Heavy wheel traffic causes soil compaction, which adversely affects crop production and may persist for several years. We applied known compaction forces to entire plots annually for 5 years, and then determined the duration of the adverse effects on the properties of a Vertisol and the performance of crops under no-till dryland cropping with residue retention. For up to 5 years after a final treatment with a 10 Mg axle load on wet soil, soil shear strength at 70-100 mm and cone index at 180-360 mm were significantly (P < 0.05) higher than in a control treatment, and soil water storage and grain yield were lower. We conclude that compaction effects persisted because (1) there were insufficient wet-dry cycles to swell and shrink the entire compacted layer, (2) soil loosening by tillage was absent and (3) there were fewer earthworms in the compacted soil. Compaction of dry soil with 6 Mg had little effect at any time, indicating that by using wheel traffic only when the soil is dry, problems can be avoided. Unfortunately such a restriction is not always possible because sowing, tillage and harvest operations often need to be done when the soil is wet. A more generally applicable solution, which also ensures timely operations, is the permanent separation of wheel zones and crop zones in the field--the practice known as controlled traffic farming. Where a compacted layer already exists, even on a clay soil, management options to hasten repair should be considered, e.g. tillage, deep ripping, sowing a ley pasture or sowing crop species more effective at repairing compacted soil.