The influence of cell surface properties of thermophilic streptococci on attachment to stainlesssteel

The quality of milk products is threatened by the formation of biofilms of thermophilicstreptococci on the internal surfaces of plate heat exchangers used in milk processing. Althoughattachment to stainless steel surfaces is one of the first stages in the development of a biofilm, themechanisms involved in attachment have not been reported. The cell surface properties of 12strains of thermophilic streptococci were examined to determine their importance in attachment tostainless steel surfaces. Hydrophobicity, extracellular polysaccharide production and cell surfacecharge varied between the different strains but could not be related to numbers attaching. Treatingthe cells with sodium metaperiodate, lysozyme or trichloroacetic acid to disrupt cell surfacepolysaccharide had no effect on attachment. Treatment with trypsin or sodium dodecyl sulphate toremove cell surface proteins resulted in a 100-fold reduction in the number of bacteria attaching.This result suggests that the surface proteins of the thermophilic streptococci are important intheir attachment to stainless steel.     

The generation and subsequent fate of glutathionyl radicals in biological systems

Horseradish peroxidase-catalyzed oxidation of p-phenetidine in the presence of either glutathione (GSH), cysteine, or N-acetylcysteine led to the production of the appropriate thioyl radical which could be observed using EPR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide. This confirms earlier work using acetaminophen (Ross, D., Albano, E., Nilsson, U., and Moldéus, P. (1984) Biochem. Biophys. Res. Commun. 125, 109-115). The further reactions of glutathionyl radicals (GS.), generated during horseradish peroxidase-catalyzed oxidation of p-phenetidine and acetaminophen in the presence of GSH, were investigated by following kinetics of oxygen uptake and oxidized glutathione (GSSG) formation. Oxygen uptake and GSSG generation were dependent on the concentration of GSH but above that which was required for maximal interaction with the primary amine or phenoxy radical generated during peroxidatic oxidation of p-phenetidine or acetaminophen, suggesting that a secondary GSH-dependent process was responsible for oxygen uptake and GSSG production. GSSG was the only product of thiol oxidation detected during peroxidatic oxidation of p-phenetidine or acetaminophen in the presence of GSH, but under nitrogen saturation conditions its production was reduced to 8 and 33% of the corresponding amounts obtained under aerobic conditions in the cases of p-phenetidine and acetaminophen, respectively. Nitrogen saturation conditions did not affect horseradish peroxidase-catalyzed metabolism. This shows that the main route of GSSG generation in such reactions is not by dimerization of GS. but via mechanism(s) involving oxygen consumption such as via GSSG-. or via GSOOH.     

Is prostaglandin E the neural mediator of the febrile response? The case against a proven obligatory role.

We have reviewed the evidence in favor of a prostaglandin mediator of the thermal responses in fever and found that PGE injected into the hypothalamus does not always cause fever, that cerebrospinal fluid concentrations of PGE are not reliable reflections of hypothalamic events, and that antipyretic drugs may act in ways other than inhibiting PGE synthesis. Fever is not blocked by prostaglandin antagonists, nor by ablation of PGE-sensitive areas of the brain. There is poor correlation between the effects of pyrogens and of PGE on cerebral neurons. There is evidence that at least one prostanoid other than prostaglandin is a mediator of fever, but the prostanoid has not been identified yet. We conclude that PGE may contribute to the neural responses in fever but is not essential.     

Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis. Purification, characterization, and physiological function

Three different deoxyribonucleoside kinases with specificities toward thymidine, deoxyguanosine, and deoxyadenosine/deoxycytidine, respectively, are identified in Bacillus subtilis. The deoxyadenosin/deoxycytidine kinase is purified 950-fold employing blue Sepharose CL-6B column chromatography. The two deoxyribonucleoside kinase activities copurify and are present in the same band after polyacrylamide gel electrophoresis. The molecular weight is determined by gel filtration to be 47,000. Cytidine, adenosine, arabinosylcytosine, and arabinosyladenine are substrates for the enzyme. The activities toward these substrates are less than 20% of the activities obtained with deoxyadenosin and deoxycytidine. The deoxycytidine and deoxyadenosine saturation curves are hyperbolic with Km values for both nucleosides around 5 microM. The maximal velocities for the two deoxyribonucleosides are nearly identical with GTP as phosphate donor. GTP is the best donor showing hyperbolic saturation curves and Km values around 150 microM depending on the deoxyribonucleoside concentration. dATP and dCTP are inhibitors when GTP is the phosphate donor. They may both act as phosphate donors themselves. A divalent metal ion is required, Mg2+ giving the highest activity. A spontaneous mutant, selected as resistant to 5-fluorodeoxycytidine, lacks both deoxycytidine and deoxyadenosine kinase activity, while it retains normal activities toward deoxyguanosine, deoxyuridine, and thymidine.     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

Biorefining of wood: combined production of ethanol and xylanase from waste fiber sludge

The possibility to utilize fiber sludge, waste fibers from pulp mills and lignocellulose-based biorefineries, for combined production of liquid biofuel and biocatalysts was investigated. Without pretreatment, fiber sludge was hydrolyzed enzymatically to monosaccharides, mainly glucose and xylose. In the first of two sequential fermentation steps, the fiber sludge hydrolysate was fermented to cellulosic ethanol with the yeast Saccharomyces cerevisiae. Although the final ethanol yields were similar, the ethanol productivity after 9.5?h was 3.3?g/l/h for the fiber sludge hydrolysate compared with only 2.2?g/l/h for a reference fermentation with similar sugar content. In the second fermentation step, the spent fiber sludge hydrolysate (the stillage obtained after distillation) was used as growth medium for recombinant Aspergillus niger expressing the xylanase-encoding Trichoderma reesei (Hypocrea jecorina) xyn2 gene. The xylanase activity obtained with the spent fiber sludge hydrolysate (8,500?nkat/ml) was higher than that obtained in a standard medium with similar monosaccharide content (1,400?nkat/ml). Analyses based on deglycosylation with N-glycosidase?F suggest that the main part of the recombinant xylanase was unglycosylated and had molecular mass of 20.7?kDa, while a minor part had N-linked glycosylation and molecular mass of 23.6?kDa. Chemical analyses of the growth medium showed that important carbon sources in the spent fiber sludge hydrolysate included xylose, small aliphatic acids, and oligosaccharides. The results show the potential of converting waste fiber sludge to liquid biofuel and enzymes as coproducts in lignocellulose-based biorefineries.