Molecular characterisation of the mechanisms of compatible solute accumulation in Listeria monocytogenes

The ability of the Gram-positive foodborne pathogen Listeria monocytogenes to survive and grow in environments of elevated osmolarity can be attributed, at least in part, to the accumulation of a restricted range of low molecular mass solutes compatible with cellular function. Accumulated to high internal concentrations in hyper-saline environments, compatible solutes, either transported into the cell or synthesized de novo, play a dual role: helping to stabilize protein structure and function while also counterbalancing external osmotic strength, thus preventing water loss from the cell and plasmolysis. While previous physiological investigations identified glycine betaine, carnitine, and proline as the principal compatible solutes in the listerial osmostress response, genetic alanysis of the uptake/synthesis systems governing the accumulation of these compounds has, until now, remained largely unexplored. Representing the first genetic analysis of compatible solute accumulation in L. monocytogenes, this thesis describes the molecular characterization of BetL; a highly specific secondary glycine betaine transport system, OpuC; a multicomponent carnitine/glycine betaine transporter, and finally proBA; a two-gene operon encoding the first two enzymes of the listerial proline piosynthesis pathway. In addition to their role in osmotolerance, the potential of each system in contributing to listerial pathogenesis was investigated. While mutations in each gene cluster exhibited dramatic reductions in listerial osmotolerance, OpuC- mutants were additionally shown to exhibit reduced virulence when admisistered via the oral route. This represents the first direct link between the salt stress response and virulence in L. monocytogenes.

Phosphorus nutrition and tolerance of cotton to water stress: II. Water relations, free and bound water and leaf expansion rate

In previous experiments, increased leaf-Phosphorus (P) content with increasing P supply enhanced the individual leaf expansion and water content of fresh cotton leaves in a severely drying soil. In this paper, we report on the bulk water content of leaves and its components, free and bound water, along with other measures of plant water status, in expanding cotton leaves of various ages in a drying soil with different P concentrations. The bound water in living tissue is more likely to play a major role in tolerance to abiotic stresses by maintaining the structural integrity and/or cell wall extensibility of the leaves, whilst an increased amount of free water might be able to enhance solute accumulation, leading to better osmotic adjustment and tolerance to water stress, and maintenance of the volumes of sub-cellular compartments for expansive leaf growth. There were strong correlations between leaf-P%, leaf water (total, free and bound water) and leaf expansion rate (LER) under water stress conditions in a severely drying soil. Increased soil-P enhanced the uptake of P from a drying soil, leading to increased supply of osmotically active inorganic solutes to the cells in growing leaves. This appears to have led to the accumulation of free water and more bound water, ultimately leading to increased leaf expansion rates as compared to plants in low P soil under similar water stress conditions. The greater amount of bound and free water in the high-P plants was not necessarily associated with changes in cell turgor, and appears to have maintained the cell-wall properties and extensibility under water stressed conditions in soils that are nutritionally P-deficient.

Acclimation to short-term low temperatures in two Eucalyptus globulus clones with contrasting drought resistance

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A Modeling Approach to Quantify the Effects of Stomatal Behavior and Mesophyll Conductance on Leaf Water Use Efficiency

Water use efficiency (WUE) is considered as a determinant of yield under stress and a component of crop drought resistance. Stomatal behavior regulates both transpiration rate and net assimilation and has been suggested to be crucial for improving crop WUE. In this work, a dynamic model was used to examine the impact of dynamic properties of stomata on WUE. The model includes sub-models of stomatal conductance dynamics, solute accumulation in the mesophyll, mesophyll water content, and water flow to the mesophyll. Using the instantaneous value of stomatal conductance, photosynthesis, and transpiration rate were simulated using a biochemical model and Penman-Monteith equation, respectively. The model was parameterized for a cucumber leaf and model outputs were evaluated using climatic data. Our simulations revealed that WUE was higher on a cloudy than a sunny day. Fast stomatal reaction to light decreased WUE during the period of increasing light (e.g., in the morning) by up to 10.2% and increased WUE during the period of decreasing light (afternoon) by up to 6.25%. Sensitivity of daily WUE to stomatal parameters and mesophyll conductance to CO2 was tested for sunny and cloudy days. Increasing mesophyll conductance to CO2 was more likely to increase WUE for all climatic conditions (up to 5.5% on the sunny day) than modifications of stomatal reaction speed to light and maximum stomatal conductance.

Electrochemical Oxidation of Hydrogen Sulfide at Platinum Electrodes in Room Temperature Ionic Liquids: Evidence for Significant Accumulation of H2S at the Pt/1-Butyl-3-methylimidazolium Trifluoromethylsulfonate Interface

Electrochemical oxidation of hydrogen sulfide gas (H2S) has been studied at a platinum microelectrode (10 mu m diameter) in five room temperature ionic liquids (RTILs): [C(4)mim][OTf], [C(4)dmim][NTf2], [C(4)mim][PF6],. [C(6)mim][FAP], and [P-14,P-6,P-6,P-6][FAP] (where [C-n mim](+) = 1-alkyl-3-methylimidazolium, [C(n)dmim](+) = 1-alkyl-2,3-dimethylimidazolium, [P-14,P-6,P-6,P-6](+) = tris(p-hexyl)-tetradecylphosphonium, [OTf](-) = trifluoromethlysulfonate, [NTf2](-) = bis(trifluoromethylsulfonyl)imide, [PF6](-) = hexafluorophosphate, and [FAP](-) = trifluorotris(pentafluoroethyl)phosphate). In four of the RTILs ([C(4)dmim][NTf2], [C(4)mim][PF6], [C(6)mim][FAP], and [P-14,P-6,P-6,P-6][FAP]), no clear oxidative signal was observed. In [C(4)mim][OTf], a chemically irreversible oxidation peak was observed on the oxidative sweep with no signal seen on the reverse scan. The oxidative signal showed an adsorptive stripping peak type followed by near steady-state limiting current behavior. Potential step chronoamperometry was carried out on the reductive wave, giving a diffusion coefficient and solubility of 1.6 x 10(-11) m(2) s(-1) and 7 mM, respectively (at 25 degrees C). Using these data, we modeled the oxidation signal kinetically, assuming adsorption preceded oxidation and that adsorption was approximately Langmuirian. The oxidation step was described by an electrochemically fully irreversible Tafel law/Butler-Volmer formalism. Modeling indicated a substantial buildup of H2S in the double layer in excess of the coverage that would be expected for a monolayer of chemisorbed H2S, reflecting high solubility of the gas in [C(4)mim][OTf] and possible attractive interactions with the [OTf](-) anions accumulated at the electrode at potentials positive of the potential of zero charge. Solute enrichment of the double layer in the solution adjacent to the electrode appears a novel feature of RTIL electrochemistry.

Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

Higher accumulation of polybrominated diphenyl ethers in infants than in adults

Pooled serum samples collected from 8132 residents in 2002/03 and 2004/05 were analyzed to assess human polybrominated diphenyl ether (PBDE) concentrations from specified strata of the Australian population. The strata were defined by age (0−4 years, 5−15 years, < 16 years, 16−30 years, 31−45 years, 46−60 years, and >60 years); region; and gender. For both time periods, infants and older children had substantially higher PBDE concentrations than adults. For samples collected in 2004/05, the mean ± standard deviation ΣPBDE (sum of the homologue groups for the mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, and deca-BDEs) concentrations for 0−4 and 5−15 years were 73 ± 7 and 29 ± 7 ng g−1 lipid, respectively, while for all adults >16 years, the mean concentration was lower at 18 ± 5 ng g−1 lipid. A similar trend was observed for the samples collected in 2002/03, with the mean ΣPBDE concentration for children <16 years being 28 ± 8 ng g−1 lipid and for the adults >16 years, 15 ± 5 ng g−1 lipid. No regional or gender specific differences were observed. Measured data were compared with a model that we developed to incorporate the primary known exposure pathways (food, air, dust, breast milk) and clearance (half-life) data. The model was used to predict PBDE concentration trends and indicated that the elevated concentrations in infants were primarily due to maternal transfer and breast milk consumption with inhalation and ingestion of dust making a comparatively lower contribution.

Changes in Level of Virus Accumulation and Incidence of Infection are Critical in the Characterization of Rice Tungro Bacilliform Virus (RTBV) Resistance in Rice

Analysis by enzyme-linked immunosorbent assay showed that Rice tungro bacilliform virus (RTBV) accumulated in a cyclic pattern from early to late stages of infection in tungro-susceptible variety, Taichung Native 1 (TN1), and resistant variety, Balimau Putih, singly infected with RTBV or co-infected with RTBV+Rice tungro spherical virus (RTSV). These changes in virus accumulation resulted in differences in RTBV levels and incidence of infection. The virus levels were expressed relative to those of the susceptible variety and the incidence of infection was assessed at different weeks after inoculation. At a particular time point, RTBV levels in TN1 or Balimau Putih singly infected with RTBV were not significantly different from the virus level in plants co-infected with RTBV+RTSV. The relative RTBV levels in Balimau Putih either singly infected with RTBV or co-infected with RTBV+RTSV were significantly lower than those in TN1. The incidence of RTBV infection varied at different times in Balimau Putih but not in TN1, and to determine the actual infection, the number of plants that became infected at least once anytime during the 4wk observation period was considered. Considering the changes in RTBV accumulation, new parameters for analyzing RTBV resistance were established. Based on these parameters, Balimau Putih was characterized having resistance to virus accumulation although the actual incidence of infection was >75%.

A two-Dimensional finite volume method for variable density flow and solute transport through saturated-unsaturated media

Extensive groundwater withdrawal has resulted in a severe seawater intrusion problem in the Gooburrum aquifers at Bundaberg, Queensland, Australia. Better management strategies can be implemented by understanding the seawater intrusion processes in those aquifers. To study the seawater intrusion process in the region, a two-dimensional density-dependent, saturated and unsaturated flow and transport computational model is used. The model consists of a coupled system of two non-linear partial differential equations. The first equation describes the flow of a variable-density fluid, and the second equation describes the transport of dissolved salt. A two-dimensional control volume finite element model is developed for simulating the seawater intrusion into the heterogeneous aquifer system at Gooburrum. The simulation results provide a realistic mechanism by which to study the convoluted transport phenomena evolving in this complex heterogeneous coastal aquifer.

The organic fraction of bubble-generated, accumulation mode Sea Spray Aerosol (SSA)

Recent studies have detected a dominant accumulation mode (~100 nm) in the Sea Spray Aerosol (SSA) number distribution. There is evidence to suggest that particles in this mode are composed primarily of organics. To investigate this hypothesis we conducted experiments on NaCl, artificial SSA and natural SSA particles with a Volatility-Hygroscopicity-Tandem-Differential-Mobility-Analyser (VH-TDMA). NaCl particles were atomiser generated and a bubble generator was constructed to produce artificial and natural SSA particles. Natural seawater samples for use in the bubble generator were collected from biologically active, terrestrially-affected coastal water in Moreton Bay, Australia. Differences in the VH-TDMA-measured volatility curves of artificial and natural SSA particles were used to investigate and quantify the organic fraction of natural SSA particles. Hygroscopic Growth Factor (HGF) data, also obtained by the VH-TDMA, were used to confirm the conclusions drawn from the volatility data. Both datasets indicated that the organic fraction of our natural SSA particles evaporated in the VH-TDMA over the temperature range 170–200°C. The organic volume fraction for 71–77 nm natural SSA particles was 8±6%. Organic volume fraction did not vary significantly with varying water residence time (40 secs to 24 hrs) in the bubble generator or SSA particle diameter in the range 38–173 nm. At room temperature we measured shape- and Kelvin-corrected HGF at 90% RH of 2.46±0.02 for NaCl, 2.35±0.02 for artifical SSA and 2.26±0.02 for natural SSA particles. Overall, these results suggest that the natural accumulation mode SSA particles produced in these experiments contained only a minor organic fraction, which had little effect on hygroscopic growth. Our measurement of 8±6% is an order of magnitude below two previous measurements of the organic fraction in SSA particles of comparable sizes. We stress that our results were obtained using coastal seawater and they can’t necessarily be applied on a regional or global ocean scale. Nevertheless, considering the order of magnitude discrepancy between this and previous studies, further research with independent measurement techniques and a variety of different seawaters is required to better quantify how much organic material is present in accumulation mode SSA.

Selective accumulation of virus-specific CD8+ T cells within the peripheral blood stem cell compartment

The absence of cellular immunity is central to the pathogenesis of herpesvirus-mediated diseases after allogeneic hemopoietic stem cell transplantation (HSCT). For both bone marrow (BM)– and granulocyte-colony stimulating factor–mobilized peripheral blood stem cells (PBSCs) HSCT, donor-derived Epstein-Barr virus (EBV) and cytomegalovirus (CMV) peptide–specific CD8+ T cells clones undergo early expansion and persist long-term, with additional diversification arising from novel antigen-specific clones from donor-derived progenitors. Whether BM or PBSC is the superior source of antiviral CD8+ T cells is unclear. Given that PBSC has largely replaced BM as a source of stem cells for HSCT, it is unlikely that herpesvirus effector T-cell reconstitution will ever be compared prospectively. PBSC grafts contain 10 to 30 times more T cells than BM and a randomized study found proven viral infections were more frequent in BM than PBSC recipients, suggesting viral-specific T-cell immunity is enhanced in PBSC. Recently Moss showed in lung cancer patients that herpesvirus-specific BM-derived CD8+ T cells have unique homing properties relative to herpesvirus-specific CD8+ T cells present in unmobilized peripheral blood (PB). Immunodominant EBV-lytic peptide–specific CD8+ T cells were enriched in BM but were reduced for CMV peptide–specific CD8+ T cells relative to PB. EBV-latent peptide–specific CD8+ T cells were equivalent, which has relevance in the context of posttransplantation lymphoproliferative disorder for which impaired EBV-latent CD8+ T-cell immunity is a risk-factor. A comparison of herpesvirus-specific cellular immunity in PBSC versus PB has yet to be performed.

Whitefly Transmission and Efficient ssDNA Accumulation of Bean Golden Mosaic Geminivirus Require Functional Coat Protein

Bean golden mosaic geminivirus (BGMV) has a bipartite genome composed of two circular ssDNA components (DNA-A and DNA-B) and is transmitted by the whitefly, Bemisia tabaci. DNA-A encodes the viral replication proteins and the coat protein. To determine the role of BGMV coat protein systemic infection and whitefly transmission, two deletions and a restriction fragment inversion were introduced into the BGMV coat protein gene. All three coat protein mutants produced systemic infections when coinoculated with DNA-B onto Phaseolus vulgaris using electric discharge particle acceleration "particle gun." However, they were not sap transmissible and coat protein was not detected in mutant-infected plants. In addition, none of the mutants were transmitted by whiteflies. With all three mutants, ssDNA accumulation of DNA-A and DNA-B was reduced 25- to 50-fold and 3- to 10-fold, respectively, as compared to that of wild-type DNA. No effect on dsDNA-A accumulation was detected and there was 2- to 5-fold increase in dsDNA-B accumulation. Recombinants between the mutated DNA-A and DNA-B forms were identified when the inoculated coat protein mutant was linearized in the common region.

Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.

Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r2 = 0.47, p < 0.001) and GAG content (r2 = 0.68, p < 0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r2 = 0.60 and 0.72 for collagen and GAG, respectively, each p < 0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate–chondrocyte constructs.