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.

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.

Helix-coil stability constants for amino acids in nonaqueous solvents. Studies of random poly(-benzyl-L-glutamate-co-L-alanine) and poly(-benzyl-L-glutamate-co-L-leucine) in a mixed solvent

The host-guest technique has been applied to the determination of the helix-coil stability constants of two naturally occurring amino acids, L-alanine and L-leucine, in a nonaqueous solvent system. Random copolymers containing L-alanine and L-leucine, respectively, as guest residues and -benzyl-L-glutamate as the host residue were synthesized. The polymers were fractionated and characterized for their amino acid content, molecular weight, and helix-coil transition behavior in a dichloroacetic acid (DCA)-1,2-dichloroethane (DCE) mixture. Two types of helix-coil transitions were carried out on the copolymers: solvent-induced transitions in DCA-DCE mixtures at 25°C and thermally induced transitions in a 82:18 (wt %) DCA-DCE mixture. The thermally induced transitions were analyzed by statistical mechanical methods to determine the Zimm-Bragg parameters, and s, of the guest residues. The experimental data indicate that, in the nonaqueous solvent, the L-alanine residue stabilizes the -helical conformation more than the L-leucine residue does. This is in contrast to their behavior in aqueous solution, where the reverse is true. The implications of this finding for the analysis of helical structures in globular proteins are discussed.

Cooking in the ungra area: Fuel efficiency, energy losses, and opportunities for reducing firewood consumption

Cooking efficiency and related fuel economy issues have been studied in a particular rural area of India. Following a description of the cooking practices and conditions in this locale, cooking efficiency is examined. A cooking efficiency of only 6% was found. The use of aluminium rather than clay pots results in an increased efficiency. In addition, cooking efficiency correlates very well with specific fuel consumption. The latter parameter is much simpler to analyse than cooking efficiency. The energy losses during cooking are examined in the second part of this case study. The major energy losses are heating of excess air, heat carried away by the combustion products, heat transmitted to the stove body and floor, and the chemical energy in charcoal residue. The energy loss due to the evaporation of cooking water is also significant because it represents about one-third of the heat reaching the pots.

An RNS based transform Architecture for H.264/AVC

This paper presents the architecture and the VHDL design of an integer 2-D DCT used in the H.264/AVC. The 2-D DCT computation is performed by exploiting it’s orthogonality and separability property. The symmetry of the forward and inverse transform is used in this implementation. To reduce the computation overhead for the addition, subtraction and multiplication operations, we analyze the suitability of carry-free position independent residue number system (RNS) for the implementation of 2-D DCT. The implementation has been carried out in VHDL for Altera FPGA. We used the negative number representation in RNS, bit width analysis of the transforms and dedicated registers present in the Logic element of the FPGA to optimize the area. The complexity and efficiency analysis show that the proposed architecture could provide higher through-put.

Effect of Crowding Agents, Signal Peptide, and Chaperone SecB on the Folding and Aggregation of E. coli Maltose Binding Protein

SecB, a soluble cytosolic chaperone component of the Secexport pathway, binds to newly synthesized precursor proteins and prevents their premature aggregation and folding and subsequently targets them to the translocation machinery on the membrane. PreMBP, the precursor form of maltose binding protein, has a 26-residue signal sequence attached to the N-terminus of MBP and is a physiological substrate of SecB. We examine the effect of macromolecular crowding and SecB on the stability and refolding of denatured preMBP and MBP. PreMBP was less stable than MBP (ΔTm =7( 0.5 K) in both crowded and uncrowded solutions. Crowding did not cause any substantial changes in the thermal stability ofMBP(ΔTm=1(0.4 K) or preMBP (ΔTm=0(0.6 K), as observed in spectroscopically monitored thermal unfolding experiments. However, both MBP and preMBP were prone to aggregation while refolding under crowded conditions. In contrast to MBP aggregates, which were amorphous, preMBP aggregates form amyloid fibrils.Under uncrowded conditions, a molar excess of SecB was able to completely prevent aggregation and promote disaggregation of preformed aggregates of MBP. When a complex of the denatured protein and SecB was preformed, SecB could completely prevent aggregation and promote folding of MBP and preMBP even in crowded solution. Thus, in addition to maintaining substrates in an unfolded, export-competent conformation, SecB also suppresses the aggregation of its substrates in the crowded intracellular environment. SecB is also able to promote passive disaggregation of macroscopic aggregates of MBP in the absence of an energy source such as ATP or additional cofactors. These experiments also demonstrate that signal peptide can reatly influence protein stability and aggregation propensity.

Fate of potassium fertilisers applied to clay soils under rainfed grain cropping in south-east Queensland, Australia

Negative potassium (K) balances in all broadacre grain cropping systems in northern Australia are resulting in a decline in the plant-available reserves of K and necessitating a closer examination of strategies to detect and respond to developing K deficiency in clay soils. Grain growers on the Red Ferrosol soils have increasingly encountered K deficiency over the last 10 years due to lower available K reserves in these soils in their native condition. However, the problem is now increasingly evident on the medium-heavy clay soils (Black and Grey Vertosols) and is made more complicated by the widespread adoption of direct drill cropping systems and the resulting strong strati. cation of available K reserves in the top 0.05-0.1 m of the soil pro. le. This paper reports glasshouse studies examining the fate of applied K fertiliser in key cropping soils of the inland Burnett region of south-east Queensland, and uses the resultant understanding of K dynamics to interpret results of field trials assessing the effectiveness of K application strategies in terms of K availability to crop plants. At similar concentrations of exchangeable K (K-exch), soil solution K concentrations and activity of K in the soil solution (AR(K)) varied by 6-7-fold between soil types. When K-exch arising from different rates of fertiliser application was expressed as a percentage of the effective cation exchange capacity (i.e. K saturation), there was evidence of greater selective adsorption of K on the exchange complex of Red Ferrosols than Black and Grey Vertosols or Brown Dermosols. Both soil solution K and AR(K) were much less responsive to increasing K-exch in the Black Vertosols; this is indicative of these soils having a high K buffer capacity (KBC). These contrasting properties have implications for the rate of diffusive supply of K to plant roots and the likely impact of K application strategies (banding v. broadcast and incorporation) on plant K uptake. Field studies investigating K application strategies (banding v. broadcasting) and the interaction with the degree of soil disturbance/mixing of different soil types are discussed in relation to K dynamics derived from glasshouse studies. Greater propensity to accumulate luxury K in crop biomass was observed in a Brown Ferrosol with a KBC lower than that of a Black Vertosol, consistent with more efficient diffusive supply to plant roots in the Ferrosol. This luxury K uptake, when combined with crops exhibiting low proportional removal of K in the harvested product (i.e. low K harvest index coarse grains and winter cereals) and residue retention, can lead to rapid re-development of stratified K profiles. There was clear evidence that some incorporation of K fertiliser into soil was required to facilitate root access and crop uptake, although there was no evidence of a need to incorporate K fertiliser any deeper than achieved by conventional disc tillage (i.e. 0.1-0.15 m). Recovery of fertiliser K applied in deep (0.25-0.3 m) bands in combination with N and P to facilitate root proliferation was quite poor in Red Ferrosols and Grey or Black Vertosols with moderate effective cation exchange capacity (ECEC, 25-35 cmol(+)/kg), was reasonable but not enough to overcome K deficiency in a Brown Dermosol (ECEC 11 cmol(+)/kg), but was quite good on a Black Vertosol (ECEC 50-60 cmol(+)/kg). Collectively, results suggest that frequent small applications of K fertiliser, preferably with some soil mixing, is an effective fertiliser application strategy on lighter clay soils with low KBC and an effective diffusive supply mechanism. Alternately, concentrated K bands and enhanced root proliferation around them may be a more effective strategy in Vertosol soils with high KBC and limited diffusive supply. Further studies to assess this hypothesis are needed.

Issues and advances in the integrated control of sheep lice.

Ongoing pressure to minimise costs of production, growing markets for low residue and organic wool and meat, resistance to chemicals in louse populations, and the deregistration of diazinon for dipping and jetting have contributed to a move away from routine annual application of lousicides to more integrated approaches to controlling lice. Advances including improved methods for monitoring and detection of lice, an expanded range of louse control products and the availability of a web-accessible suite of decision support tools for wool growers (LiceBossTM) will aid this transition. Possibilities for the future include an on-farm detection test and non-chemical control methods. The design and extension of well-constructed resistance management programs to preserve the effectiveness of recently available new product groups should be a priority.

Selectivity of chlorantraniliprole to parasitoid wasps.

BACKGROUND: Chlorantraniliprole is a novel anthranilic diamide insecticide registered for use in vegetables, fruits, grains and turf against a variety of insect pests. The objective of this article is to summarize results of acute toxicity testing of chlorantraniliprole on seven species of parasitic wasps with wide geographic distribution and relevance to different crops and integrated pest management (IPM) programmes. RESULTS: Tier-1, worst-case laboratory studies evaluated wasp survival and reproduction following different exposure concentrations and scenarios to chlorantraniliprole (i.e. fresh-dried spray deposits on glass plates, direct contact, ingestion, egg card, dipped leaf residue bioassays, sprayed mummies). No statistically significant effects on adult survival, percentage parasitism or emergence were observed following exposures to chlorantraniliprole compared with controls. CONCLUSION: Chlorantraniliprole was harmless to the parasitoid wasp species tested according to IOBC classification criteria (<30% effects) and may be a useful tool in IPM programmes.

New Modified Dipping Method Using 4-hexylresorcinol for Preventing Blackspot Formation in Prawns.

Industry acceptance of alternative sulphite treatments for preventing blackspot in prawns is limited. This study demonstrated that dipping prawns (Penaeus monodon and Fenneropenaeus merguiensis) in a less concentrated solution for longer times provides better control of blackspot formation than the recommended 2 minute dip in 50 mg/L 4-hexylresorcinol. Protection improved as modified dipping times and solution concentrations increased. Blackspot protection provided by most of the modified dip methods was more effective than a 1 minute dip in 1% sodium metabisulphite. The recommended 2 minute dip method should be limited to 125 kg when used on consecutive 25-kg batches of prawns. Yields increased by less than 10% even after 72-hour exposure in the modified dip. The new dipping method meets most international residue standards when applying the modified treatments evaluated.

A novel approach to structure—flammability correlation in polyphosphate esters

The relationship between the structure and flammability of a number of polyphosphate esters has been examined. The conventional correlation of char residue with limiting oxygen index was found to be unproductive in these polymers, giving insight into the importance of gas-phase reactions in addition to condensed-phase reactions in determining their flammability. A novel approach was sought in understanding the structure-flammability relationships of these polymers relating thermal stability, phosphorus content and limiting oxygen index. An empirical relationship has been derived amongst these three parameters.

Function and regulation of Cdk7

Understanding the process of cell division is crucial for modern cancer medicine due to the central role of uncontrolled cell division in this disease. Cancer involves unrestrained proliferation as a result of cells loosing normal control and being driven through the cell cycle, where they normally would be non-dividing or quiescent. Progression through the cell cycle is thought to be dependent on the sequential activation of cyclin-dependent kinases (Cdks). The full activation of Cdks requires the phosphorylation of a conserved residue (threonine-160 on human Cdk2) on the T-loop of the kinase domain. In metazoan species, a trimeric complex consisting of Cdk7, cyclin H and Mat1 has been suggested to be the T-loop kinase of several Cdks. In addition, Cdk7 have also been implicated in the regulation of transcription. Cdk7, cyclin H, and Mat1 can be found as subunits of general transcription factor TFIIH. Cdk7, in this context, phosphorylates the Carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNA pol II), specifically on serine-5 residues of the CTD repeat. The regulation of Cdk7 in these and other functions is not well known and the unambiguous characterization of the in vivo role of Cdk7 in both T-loop activation and CTD serine-5 phosphorylation has proved challenging. In this study, the fission yeast Cdk7-cyclin H homologous complex, Mcs6-Mcs2, is identified as the in vivo T-loop kinase of Cdk1(Cdc2). It also identifies multiple levels of regulation of Mcs6 kinase activity, i.e. association with Pmh1, a novel fission yeast protein that is the apparent homolog of metazoan Mat1, and T-loop phosphorylation of Mcs6, mediated by Csk1, a monomeric T-loop kinase with similarity to Cak1 of budding yeast. In addition, Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase is identified by its interactions with Mcs2 and Pmh1. The Skp1 association with Mcs2 and Pmh1 is however SCF independent and does not involve proteolytic degradation but may reflect a novel mechanism to modulate the activity or complex assembly of Mcs6. In addition to Cdk7, also Cdk8 has been shown to have CTD serine-5 kinase activity in vitro. Cdk8 is not essential in yeast but has been shown to function as a transcriptional regulator. The function of Cdk8 is unknown in flies and mammals. This prompted the investigation of murine Cdk8 and its potential role as a redundant CTD serine-5 kinase. We find that Cdk8 is required for development prior to implantation, at a time that is co-incident with a burst of Cdk8 expression during normal development. The results does not support a role of Cdk8 as a serine-5 CTD kinase in vivo but rather shows an unexpected requirement for Cdk8, early in mammalian development. The results presented in this thesis extends our current knowledge of the regulation of the cell cycle by characterizing the function of two distinct cell cycle regulating T-loop kinases, including the unambiguous identification of Mcs6, the fission yeast Cdk7 homolog, as the T-loop kinase of Cdk1. The results also indicate that the function of Mcs6 is conserved from fission yeast to human Cdk7 and suggests novel mechanisms by which the distinct functions of Cdk7 and Mcs6 could be regulated. These findings are important for our understanding of how progression of the cell cycle and proper transcription is controlled, during normal development and tissue homeostasis but also under condition where cells have escaped these control mechanisms e.g. cancer.

Structural effects of a dimer interface mutation on catalytic activity of triosephosphate isomerase.The role of conserved residues and complementary mutations

The active site of triosephosphate isomerase (TIM, EC: 5.3.1.1), a dimeric enzyme, lies very close to the subunit interface. Attempts to engineer monomeric enzymes have yielded well-folded proteins with dramatically reduced activity. The role of dimer interface residues in the stability and activity of the Plasmodium falciparum enzyme, PfTIM, has been probed by analysis of mutational effects at residue 74. The PfTIM triple mutant W11F/W168F/Y74W (Y74W*) has been shown to dissociate at low protein concentrations, and exhibits considerably reduced stability in the presence of denaturants, urea and guanidinium chloride. The Y74W* mutant exhibits concentration-dependent activity, with an approximately 22-fold enhancement of kcat over a concentration range of 2.5–40 μm, suggesting that dimerization is obligatory for enzyme activity. The Y74W* mutant shows an approximately 20-fold reduction in activity compared to the control enzyme (PfTIM WT*, W11F/W168F). Careful inspection of the available crystal structures of the enzyme, together with 412 unique protein sequences, revealed the importance of conserved residues in the vicinity of the active site that serve to position the functional K12 residue. The network of key interactions spans the interacting subunits. The Y74W* mutation can perturb orientations of the active site residues, due to steric clashes with proximal aromatic residues in PfTIM. The available crystal structures of the enzyme from Giardia lamblia, which contains a Trp residue at the structurally equivalent position, establishes the need for complementary mutations and maintenance of weak interactions in order to accommodate the bulky side chain and preserve active site integrity.