Stimulation of thaumarchaeal ammonia oxidation by ammonia derived from organic nitrogen but not added inorganic nitrogen

Ammonia oxidation, the first step in nitrification, is performed by autotrophic bacteria and thaumarchaea, whose relative contributions vary in different soils. Distinctive environmental niches for the two groups have not been identified, but evidence from previous studies suggests that activity of thaumarchaea, unlike that of bacterial ammonia oxidizers, is unaffected by addition of inorganic N fertilizer and that they preferentially utilize ammonia generated from the mineralization of organic N. This hypothesis was tested by determining the influence of both inorganic and organic N sources on nitrification rate and ammonia oxidizer growth and community structure in microcosms containing acidic, forest soil in which ammonia oxidation was dominated by thaumarchaea. Nitrification rate was unaffected by the incubation of soil with inorganic ammonium but was significantly stimulated by the addition of organic N. Oxidation of ammonia generated from native soil organic matter or added organic N, but not added inorganic N, was accompanied by increases in abundance of the thaumarchaeal amoA gene, a functional gene for ammonia oxidation, but changes in community structure were not observed. Bacterial amoA genes could not be detected. Ammonia oxidation was completely inhibited by 0.01% acetylene in all treatments, indicating ammonia monooxygenase-dependent activity. The findings have implications for current models of soil nitrification and for nitrification control strategies to minimize fertilizer loss and nitrous oxide production.     

Biological treatment of eucalypt spent sulphite liquors: a way to boost the production of second generation bioethanol

The fermentation of reducing sugars from hardwood (eucalypt) spent sulphite liquor (HSSL) into ethanol by Pichia (Scheffersomyces) stipitis is hindered by concomitant inhibitors of microbial metabolism. The conditions for the HSSL biological treatment step by Paecilomyces variotii were evaluated and optimised. Two different strategies of reactor operation were compared using single batch (B) and sequential batch reactor (SBR). Biological treatment of HSSL in the SBR revealed the best results with respect to the removal of microbial inhibitors. Also, most of inhibitory compounds, acetic acid, gallic acid, pyrogallol, amongst others, were removed from HSSL by P. variotii before the ethanol fermentation. The bio-detoxified HSSL was subjected to a successful fermentation by P. stipitis, attaining a maximum ethanol concentration of 2.4 g L⁻¹ with a yield of 0.24 g ethanol g sugars⁻¹.     

Regulation of cathepsins S and L by cystatin F during maturation of dendritic cells

In dendritic cells (DCs) cysteine cathepsins play a key role in antigen processing, invariant chain (Ii) cleavage and regulation of cell adhesion after maturation stimuli. Cystatin F, a cysteine protease inhibitor, is present in DCs in endosomal/lysosomal vesicles and thus has a potential to modulate cathepsin activity. In immature DCs cystatin F colocalizes with cathepsin S. After induction of DC maturation however, it is translocated into lysosomes and colocalizes with cathepsin L. The inhibitory potential of cystatin F depends on the properties of the monomer. We showed that the full-length monomeric cystatin F was a 12-fold stronger inhibitor of cathepsin S than the N-terminally processed cystatin F, whereas no significant difference in inhibition was observed for cathepsins L, H and X. Therefore, the role of cystatin F in regulating the main cathepsin S function in DCs, i.e. the processing of Ii, may depend on the form of the monomer present in endosomal/lysosomal vesicles. On the other hand, intact and truncated monomeric cystatin F are both potent inhibitors of cathepsin L and it is likely that cystatin F could regulate its activity in maturing, adherent DCs, controlling the processing of procathepsin X, which promotes cell adhesion via activation of Mac-1 (CD11b/CD18) integrin receptor.     

Transport and bioactivity of cyanidin 3-glucoside into the vascular endothelium

Flavonoids are dietary components involved in decreasing oxidative stress in the vascular endothelium and thus the risk of endothelial dysfunction. However, their very low concentrations in plasma place this role in doubt. Thus, a relationship between the effective intracellular concentration of flavonoids and their bioactivity needs to be assessed. This study examined the uptake of physiological concentrations of cyanidin 3-glucoside, a widespread dietary flavonoid, into human vascular endothelial cells. Furthermore, the involvement of the membrane transporter bilitranslocase (TC No. 2.A.65.1.1) as the key underlying molecular mechanism for membrane transport was investigated by using purified anti-sequence antibodies binding at the extracellular domain of the protein. The experimental observations were carried out in isolated plasma membrane vesicles and intact endothelial cells from human endothelial cells (EA.hy926) and on an ischemia-reperfusion model in isolated rat hearts. Cyanidin 3-glucoside was transported via bilitranslocase into endothelial cells, where it acted as a powerful intracellular antioxidant and a cardioprotective agent in the reperfusion phase after ischemia. These findings suggest that dietary flavonoids, despite their limited oral bioavailability and very low postabsorption plasma concentrations, may provide protection against oxidative stress-based cardiovascular diseases. Bilitranslocase, by mediating the cellular uptake of some flavonoids, is thus a key factor in their protective activity on endothelial function.     

The influence of Na+ ions on histamine secretion from mast cells induced by NGF or compound 48/80

It has been demonstrated that the release of histamine from mast cells by cytokines is strongly dependent on extracellular Ca2+ and Na+ ions. The results of our current research indicate that the removal of extracellular Na+ enhances NGF induced histamine release, but reduces induction by compound 48/80, suggesting that different mechanisms are involved in the secretory process induced by these agents.     

Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells

Background

We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments.

Results

K14 IBs were electroporated into SW13 cells grown in culture together with a ??reporter?? plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5.

Conclusions

Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.     

Dorsal longitudinal stretch receptor of Drosophila melanogaster larva - fine structure and maturation

Insects possess two types of sensory neurons: ciliated type I sensory neurons that innervate external sensory organs and chordotonal organs, and type II sensory neurons that form a subepidermal plexus or innervate stretch receptors. Among stretch receptors, a dorsel longitudinal stretch receptor is highly conserved in insects, being found in all insect orders investigated. Here we describe the topology and anatomical structure of this receptor in the fruit fly embryo and larva using transmission electron microscopy and single cell staining for fluorescence microscopy. The receptor is composed of the dorsal bipolar dendrite neuron, which arises from an archetypal cell lineage, its sister glial cell and the peripheral glial cell accompanying the nerve. The neuron is situated among the muscles in the dorsal body wall on the intersegmental nerve. Its two dendrites stretch the length of the segment to the segmental folds. The neuron is wrapped by both glial cells and surrounded by a common basal lamina, which fans out at the dendritic tips to attach them to the epidermal cells at the segmental borders.     

Finding differentially expressed genes in two-channel DNA microarray datasets: how to increase reliability of data preprocessing

Due to the great variety of preprocessing tools in two-channel expression microarray data analysis it is difficult to choose the most appropriate one for a given experimental setup. In our study, two independent two-channel inhouse microarray experiments as well as a publicly available dataset were used to investigate the influence of the selection of preprocessing methods (background correction, normalization, and duplicate spots correlation calculation) on the discovery of differentially expressed genes. Here we are showing that both the list of differentially expressed genes and the expression values of selected genes depend significantly on the preprocessing approach applied. The choice of normalization method to be used had the highest impact on the results. We propose a simple but efficient approach to increase the reliability of obtained results, where two normalization methods which are theoretically distinct from one another are used on the same dataset. Then the intersection of results, that is, the lists of differentially expressed genes, is used in order to get a more accurate estimation of the genes that were de facto differentially expressed.