Inhibition of Methanosarcina barkeri strain 227 by cadmium

Abstract The effect of cadmium (Cd) on methane formation from methanol and/or H₂–CO₂ by Methanosarcina barkeri was examined in a defined growth medium and in a simplified buffer system containing 50 mM Tes with or without 2 mM dithiothreitol (DTT). No inhibition of methanogenesis by high concentrations of cadmium was observed in growth medium. Similarly, little inhibition of methanogenesis by whole cells in the Tes buffer system was observed in the presence of 430 μM Cd or 370 μM mercury (Hg) with 2 mM DTT. When the concentration of DTT was reduced to 0.4 mM, almost complete inhibition of methanogenesis from H₂–CO₂ and methanol by 600 μM Cd was observed. In the absence of DTT, 150 μM Cd inhibited methanogenesis from H₂–CO₂ completely and from methanol by 97%. Methanogenesis from H₂–CO₂ was more sensitive to Cd than that from methanol.     

Effect of pulmonary oedema induced by alpha-naphthylthiourea on synthesis of cyclo-oxygenase products in rat isolated lungs

Synthesis of COP (prostaglandins; PG and thromboxanes; Tx) from exogenous and endogenous arachidonic acid (AA) was studied in isolated perfused lungs from rats treated in vivo with a single dose of alpha-naphthylthiourea (ANTU; 10mg/kg;). Lung dry:wet weight ratios showed changes characteristic of oedema between 6 and 16h after ANTU. Bioassay of COP showed that COP synthesis from exogenous AA was raised above control values in lungs from rats treated with ANTU, reaching a maximum at 16h after treatment. By radioimmunoassay, the major increase was in 6-oxo-PGF1 alpha, with lesser effects on PGE2 and PGF2 alpha levels. Synthesis of bioassayable COP from endogenous AA induced by the calcium ionophore A23187 was increased as early as 2h after ANTU treatment and remained elevated up to 70h. In lungs 28h after ANTU, 6-oxo-PGF1 alpha release was greater than in normal lungs. These results show that in this model of pulmonary oedema, the potential for COP synthesis was increased. From the time course of this effect, increased COP synthesis was probably a response to the initial damage rather than a cause of the oedema.     

Effect of pulmonary oedema induced by α-naphthylthiourea on synthesis of cyclo-oxygenase products in rat isolated lungs

Synthesis of COP (prostaglandins; PG and thromboxanes; Tx) from exogenous and endogenous arachidonic acid (AA) was studied in isolated perfused lungs from rats treated in vivo with a single dose of α-naphthylthiourea (ANTU; 10mg/kg;). Lung dry: wet weight ratios showed changes characteristic of oedema between 6 and 16h after ANTU. Bioassay of COP showed that COP synthesis from exogenous AA was raised above control values in lungs from rats treated with ANTU, reaching a maximum at 16h after treatment. By radioimmunoassay, the major increase was in 6-oxo-PGF_1α, with lesser effects on PGE₂ and PGF_2α levels. Synthesis of bioassayable COP from endogenous AA induced by the calcium ionophore A23187 was increased as early as 2h after ANTU treatment and remained elevated up to 70h. In lungs 28h after ANTU, 6-oxo-PGF_1α release was greater than in normal lungs. These results show that in this model of pulmonary oedema, the potential for COP synthesis was increased. From the time course of this effect, increased COP synthesis was probably a response to the initial damage rather than a cause of the oedema.     

Extraction of periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough)

Abstract Periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) was extracted according to the method of van der Westen [8] and the effect of trace minerals on the extractability of this enzyme was investigated. The final growth yields in the presence or absence of trace minerals were the same; however, the growth was much faster and the amount of periplasmic hydrogenase extracted was significantly lower in the presence of trace minerals. Polyacrylamide gel electrophoresis showed the presence of 2 hydrogenases in D. vulgaris , one soluble and the other possibly membrane-bound.     

Lactate conversion to acetate,CO2 and H2 in cell suspensions of Desulfovibrio vulgaris (Marburg): indications for the involvement of an energy driven reaction

Cell suspensions of Desulfovibrio vulgaris were found to catalyze, in the absence of sulfate, the complete conversion of 1 lactate to 1 acetate, 1 CO₂, and 2 H₂ (G₀=-8.8 kJ/mol) and of 1 pyruvate to 1 acetate, 1 CO₂, and 1 H₂ (G₀=-52 kJ/mol). Protonophores, the proton translocating ATPase inhibitor N,N-dicyclohexylcarbodiimide, and arsenate specifically inhibited H₂ formation from lactate but not from pyruvate. The results suggest that lactate oxidation to pyruvate and H₂ (G ₀=+43.2 kJ/mol) is energy driven.     

Heavy metal inhibition of methanogenesis by Methanospirillum hungatei GP1

Abstract Washed whole cells of Methanospirillum hungatei incubated in TES buffer retained methanogenic activity in the absence of any reducing agents. Washed cells grown with 80% H₂-20% CO₂ and acetate produced methane from H₂/CO₂ and 50 mM formate at 1.1 to 1.8 and 15 μmol methane · h⁻¹· mg⁻¹ protein, respectively. Cadmium at a concentration of 15 μM and 50 μM mercury, copper or zinc completely inhibited methane production from H₂/CO₂ by M. hungatei . The chelating agent, EDTA, protected the cells from inhibition by cadmium but acetate and citrate did not. The activity of formate dehydrogenase and hydrogenase remaining in cells after incubation with copper, mercury, zinc or cadmium was reduced with formate dehydrogenase being the more sensitive.     

Systematic Mutagenesis of Genes Encoding Predicted Autotransported Proteins of Burkholderia pseudomallei Identifies Factors Mediating Virulence in Mice,Net Intracellular Replication and a Novel Protein Conferring Serum Resistance

Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log₁₀ compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.