Evidence for a new extracellular peroxidase. Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55.

A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp. BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named 'Manganese-Inhibited Peroxidase'.     

Fed-batch operation of an industrial cell culture process in shaken microwells

Recently we have demonstrated batch suspension culture of mammalian cells in microwell plates. Here we describe a method for fed-batch culture of an industrially relevant GS-CHO (Glutamine Synthetase-Chinese Hamster Ovary) cell line in shaken 24-standard round well (24-SRW) plates. Use of a commercially available ‘sandwich lid’ and appropriate dilution of the bolus feeds counteracted liquid evaporation from the wells resulting in similar cell growth and antibody formation kinetics in both 24-SRW plates (800 μl) and shaken flasks (50 ml). Peak viable cell densities obtained were 8 ± 0.5 × 10⁶ and 9 ± 1.3 × 10⁶ ml⁻¹, respectively, while comparable final titres of a whole IgG of approximately 1.5 g l⁻¹ were recorded. Use of microwells provides at least a 50-fold reduction in medium requirements compared to shake-flask and other culture devices currently used in early stage cell culture process development. The ability to run multiple wells in parallel and to automate culture operation also offers considerable enhancements in experimental throughput.     

A role for IL-15 in the migration of effector CD8 T cells to the lung airways following influenza infection

The cytokines generated locally in response to infection play an important role in CD8 T cell trafficking, survival, and effector function, rendering these signals prime candidates for immune intervention. In this paper, we show that localized increases in the homeostatic cytokine IL-15 induced by influenza infection is responsible for the migration of CD8 effector T cells to the site of infection. Moreover, intranasal delivery of IL-15-IL-15Rα soluble complexes (IL-15c) specifically restores the frequency of effector T cells lost in the lung airways of IL-15-deficient animals after influenza infection. Exogenous IL-15c quantitatively augments the respiratory CD8 T cell response, and continued administration of IL-15c throughout the contraction phase of the anti-influenza CD8 T cell response magnifies the resultant CD8 T cell memory generated in situ. This treatment extends the ability of these cells to protect against heterologous infection, immunity that typically depreciates over time. Overall, our studies describe what to our knowledge is a new function for IL-15 in attracting effector CD8 T cells to the lung airways and suggest that adjuvanting IL-15 could be used to prolong anti-influenza CD8 T cell responses at mucosal surfaces to facilitate pathogen elimination.     

Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications

Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences--the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The 'environmental packages' apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.     

Investigation of the potential for triterpene synthesis in rice through genome mining and metabolic engineering

The first committed step in sterol biosynthesis in plants involves the cyclization of 2,3-oxidosqualene by the oxidosqualene cyclase (OSC) enzyme cycloartenol synthase. 2,3-Oxidosqualene is also a precursor for triterpene synthesis. Antimicrobial triterpenes are common in dicots, but seldom found in monocots, with the notable exception of oat. Here, through genome mining and metabolic engineering, we investigate the potential for triterpene synthesis in rice. The first two steps in the oat triterpene pathway are catalysed by a divergent OSC (AsbAS1) and a cytochrome P450 (CYP51). The genes for these enzymes form part of a metabolic gene cluster. To investigate the origins of triterpene synthesis in monocots, we analysed systematically the OSC and CYP51 gene families in rice. We also engineered rice for elevated triterpene content. We discovered a total of 12 OSC and 12 CYP51 genes in rice and uncovered key events in the evolution of triterpene synthesis. We further showed that the expression of AsbAS1 in rice leads to the accumulation of the simple triterpene, β-amyrin. These findings provide new insights into the evolution of triterpene synthesis in monocots and open up opportunities for metabolic engineering for disease resistance in rice and other cereals.     

Exposure of dog thyroid slices to acetylcholine induces refractoriness to its subsequent stimulation of glucose oxidation

An initial incubation of dog thyroid slices with 0.1 or 1 microM acetylcholine (ACH) for at least 2 h decreases its subsequent stimulation of [1-14C]glucose oxidation. Refractoriness persists for as long as 6 h in the absence of ACH. While new protein synthesis is essential for recovery, it is not necessary for its induction. Refractoriness is prevented when 25 microM tropicamide, an atropine-like drug, is present from the beginning of the initial incubation, but not when it is added after 2 h of incubation of slices with ACH, indicating that at this time ACH is no longer necessary for refractoriness. During refractoriness induced by ACH, stimulation of glucose oxidation by thyroid-stimulating hormone, prostaglandin E1, dibutyryl cyclic AMP, and cholera toxin, but not menadiol, is also significantly diminished. Incubation of thyroid slices with ACH does not modify its stimulation of iodide organification or 32Pi incorporation into phospholipids. These results suggest that the desensitization is not due to changes in the ACH receptor but rather to intracellular metabolic effects. This phenomenon may be important in the regulation of cholinergic effects on the thyroid.     

Osmotic water permeability of small intestinal brush-border membranes

Summary A stopped-flow nephelometric technique was used to examine osmotic water flow across small intestinal brush-border membranes. Brush-border membrane vesicles (BBMV) were prepared from rat small intestine by calcium precipitation. Scattered 500 nm light intensity at 90° to incident was a linear function of the number of vesicles in suspension, and of the reciprocal of the suspending medium osmolality. When BBMV were mixed with hyperosmotic mannitol solutions there was a rapid increase in the intensity of scattered light that could be fit to a single exponential function. The rate constant for vesicle shrinking varied with temperature and the size of the imposed osmotic gradient. At 25°C and an initial osmotic gradient of 50 mOsm, the rate constant was 1.43±0.044 sec^–1. An Arrhenius plot of the temperature dependence of vesicle shrinking showed a break at about 25°C with an activation energy of 9.75±1.04 kcal/mole from 11 to 25°C and 17.2±0.55 kcal/mole from 25 to 37°C. The pore-forming antibiotic gramicidin increased the rate of osmotically driven water efflux and decreased the activation energy of the process to 4.51±0.25 kcal/mole. Gramicidin also increased the sodium permeability of these membranes as measured by the rate of vesicle reswelling in hyperosmotic NaSCN medium. Gramicidin had no effect on mannitol permeability. Assuming spherical vesicles of 0.1 m radius, an osmotic permeability coefficient of 1.2×10^–3 cm/sec can be estimated for the native brush-border membranes at 25°C. These fesults are consistent with the solubility-diffusion model for water flow across small intestinal BBMV but are inconsistent with the existence there of large aqueous pores.     

Diversity of European habitat types is correlated with geography more than climate and human pressure

Habitat richness, that is, the diversity of ecosystem types, is a complex, spatially explicit aspect of biodiversity, which is affected by bioclimatic, geographic, and anthropogenic variables. The distribution of habitat types is a key component for understanding broad‐scale biodiversity and for developing conservation strategies. We used data on the distribution of European Union (EU) habitats to answer the following questions: (i) how do bioclimatic, geographic, and anthropogenic variables affect habitat richness? (ii) Which of those factors is the most important? (iii) How do interactions among these variables influence habitat richness and which combinations produce the strongest interactions? The distribution maps of 222 terrestrial habitat types as defined by the Natura 2000 network were used to calculate habitat richness for the 10 km × 10 km EU grid map. We then investigated how environmental variables affect habitat richness, using generalized linear models, generalized additive models, and boosted regression trees. The main factors associated with habitat richness were geographic variables, with negative relationships observed for both latitude and longitude, and a positive relationship for terrain ruggedness. Bioclimatic variables played a secondary role, with habitat richness increasing slightly with annual mean temperature and overall annual precipitation. We also found an interaction between anthropogenic variables, with the combination of increased landscape fragmentation and increased population density strongly decreasing habitat richness. This is the first attempt to disentangle spatial patterns of habitat richness at the continental scale, as a key tool for protecting biodiversity. The number of European habitats is related to geography more than climate and human pressure, reflecting a major component of biogeographical patterns similar to the drivers observed at the species level. The interaction between anthropogenic variables highlights the need for coordinated, continental‐scale management plans for biodiversity conservation.