Aqueous two-phase systems extraction of alpha-toxin from Clostridium perfringens type A

Two sequential half-fraction designs were applied to studying the alpha-toxin partition produced by Clostridium perfringens type A in aqueous two phase systems (ATPS), as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows, in a single step, an alpha-toxin purification of 4.6-fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase.     

Pressure affects the structure and the dynamics of the D-galactose/D-glucose-binding protein from Escherichia coli by perturbing the C-terminal domain of the protein

The effect of the pressure on the structure and stability of the D-galactose/D-glucose binding protein from Escherichia coli in the absence (GGBP) and in the presence (GGBP/Glc) of glucose was studied by Fourier transform infrared (FT-IR) spectroscopy and molecular dynamic (MD) simulations. FT-IR spectroscopy experiments showed that the protein beta-structures are more resistant than alpha-helices structures to pressure value increases. In addition, the infrared data indicated that the binding of glucose stabilizes the protein structure against high pressure values, and the protein structure does not completely unfold up to pressure values close to 9000 bar. MD simulations allow a prediction of the most probable configuration of the protein, consistent with the increasing pressures on the two systems. The detailed analysis of the structures at molecular level confirms that, among secondary structures, alpha-helices are more sensitive than beta-structures to the destabilizing effect of high pressure and that glucose is able to preserve the structure of the protein in the complex. Moreover, the evidence of the different resistance of the two domains of this protein to high pressure is investigated and explained at a molecular level, indicating the importance of aromatic amino acid in protein stabilization.     

Modulation of GJA1 turnover and intercellular communication by proinflammatory cytokines in the anterior pituitary folliculostellate cell line TtT/GF

Our previous studies have advanced the idea that the folliculostellate cell GJA1 (gap junction membrane channel protein alpha1; previously known as connexin 43)-mediated gap junctions contribute to the establishment of an intercellular network that regulates the paracrine messages and the endocrine response within the anterior pituitary. The folliculostellate cells are targets for growth factors and cytokines that modulate hormone secretion. Proinflammatory cytokines modulate the cell-to-cell communication in many tissues of the body. The present study measured the effect of the proinflammatory cytokines tumor necrosis factor and interleukin-1 on the GJA1-mediated intercellular communication, specifically the expression, localization, degradation, and phosphorylation status of GJA1 in the folliculostellate cell line TtT/GF. The GJA1 localized to the plasma membrane and to minute cytoplasmic vesicles in the perinuclear area. Using different antibodies that recognize distinctly the nonphosphorylated from the phosphorylated forms of GJA1, we showed that nonphosphorylated GJA1 in Ser-368 (NP-GJA1) localized chiefly in the cytoplasm, whereas GJA1 phosphorylated in Ser-368 (P-GJA1) localized to the plasma membrane in controls. The cytokine treatment transiently increased 1) GJA1, NP-GJA1, and P-GJA1 levels; 2) NP-GJA1 and P-GJA1 degradation by both the lysosomal and proteasomal pathways; and 3) cell-to-cell communication in TtT/GF cells. The results suggest that the cytokine-evoked, transient enhancement of folliculostellate cell-mediated intercellular communication contributes to the coordination of the response among folliculostellate cells.     

The expression of nifB gene from Herbaspirillum seropedicae is dependent upon the NifA and RpoN proteins

The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a -24/-12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of nifB::lacZ occurred only in the presence of functional rpoN and Klebsiella pneumoniae nifA genes. In addition, the integration host factor (IHF) stimulated the expression of the nifB::lacZ fusion in this background. In H. seropedicae, nifB expression occurred only in the absence of ammonium and under low levels of oxygen, and it was shown to be strictly dependent on NifA. DNA band shift experiments showed that purified K. pneumoniae RpoN and E. coli IHF proteins were capable of binding to the nifB promoter region, and in vivo dimethylsulfate footprinting showed that NifA binds to both NifA-binding sites. These results strongly suggest that the expression of the nifB promoter of H. seropedicae is dependent on the NifA and RpoN proteins and that the IHF protein stimulates NifA activation of nifB promoter.     

Recent advances in understanding of the DNA double-strand break repair machinery of plants

Living cells suffer numerous and varied alterations of their genetic material. Of these, the DNA double-strand break (DSB) is both particularly threatening and common. Double-strand breaks arise from exposure to DNA damaging agents, but also from cell metabolism-in a fortuitous manner during DNA replication or repair of other kinds of lesions and in a programmed manner, for example during meiosis or V(D)J gene rearrangement. Cells possess several overlapping repair pathways to deal with these breaks, generally designated as genetic recombination. Genetic and biochemical studies have provided considerable amounts of data about the proteins involved in recombination processes and their functions within these processes. Although they have long played a key role in building understanding of genetics, relatively little is known at the molecular level of the genetic recombination processes in plants. The use of reverse genetic approaches and the public availability of sequence tagged mutants in Arabidopsis thaliana have led to increasingly rapid progress in this field over recent years. The rapid progress of studies of recombination in plants is obviously not limited to the DSB repair machinery as such and we ask readers to understand that in order to maintain the focus and to rest within a reasonable length, we present only limited discussion of the exciting advances in the of plant meiosis field, which require a full review in their own right . We thus present here an update on recent advances in understanding of the DSB repair machinery of plants, focussing on Arabidopsis and making a particular effort to place these in the context of more general of understanding of these processes.     

Bromodeoxyuridine labelling and fluorescence‐activated cell sorting of polyamine‐transforming bacterioplankton in coastal seawater

Polyamines (PAs) are a group of nitrogen‐rich dissolved organic nitrogen (DON) compounds that are ubiquitously distributed in marine environments. To identify bacteria that are involved in PA transformations, coastal bacterioplankton microcosms were amended with a single PA model compound, i.e. putrescine (PUT) or spermidine (SPD), or with no addition as controls (CTRs). Bromodeoxyuridine (BrdU) was added to all the microcosms to label newly synthesized DNAs. Fluorescence‐activated cell sorting (FACS) analysis indicated significant increases in numbers of total cells and cells with both high and low levels of BrdU incorporation in the PUT and SPD microcosms, but not in the CTRs. 16S rDNA pyrotag sequencing of FACS‐sorted cells indicated that PUT‐ and SPD‐transforming bacteria were composed similarly of a diverse group of taxa affiliated with Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria (especially Roseobacter of its alpha lineage). Broad taxonomic distribution of PA‐transforming bacteria was also indicated by the abundance and distribution of PA transporter gene homologues in a survey of sequenced marine bacterial genomes. Our results suggest that PAs may be common DON substrates for marine bacterioplankton, in line with the hypothesis that bacterially mediated PA transformation accounts for an important proportion of marine DON flux.     

Affinity prefractionation for MS‐based plasma proteomics

The plasma proteome has proven to be one of the most challenging proteomes to profile using currently available proteomics technologies. A plethora of methodologies have been used to profile human plasma in order to discover potential biomarkers for disease and for therapy optimization. Affinity‐based prefractionation coupled to MS has been shown to be one of the most successful ways to dig deeper into the plasma proteome. Depletion of high abundant plasma proteins is becoming an initial method of choice in any plasma profiling project. However, several other affinity‐based enrichment methods have been published in recent years. Here we review both protein and peptide affinity prefractionation methods coupled with MS‐based proteomics. Analysis of the proportion of cellular and extracellular annotated proteins of publicly available MS plasma proteomics data is performed to estimate the analytical depth of various prefractionation methods.     

Pre-adipocytes commitment to neurogenesis 1: Preliminary localisation of cholinergic molecules

A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimer's disease and motor neuron disease). A stem cell population (i.e. pre-adipocytes) is present in the adipose stromal compartment. Pre-adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre-adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre-adipocytes, in parallel with neural morphology. The passage of the choline-acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic-like phenotype from pre-adipocytes.