BioVyon Protein A, an alternative solid-phase affinity matrix for chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) is an important technique in the study of DNA/protein interactions. The ChIP procedure, however, has limitations in that it is lengthy, can be inconsistent, and is prone to nonspecific binding of DNA and proteins to the bead-based solid-phase matrices that are often used for the immunoprecipitation step. In this investigation, we examined the utility of a new matrix for ChIP assays, BioVyon Protein A, a solid support based on porous polyethylene. In ChIP experiments carried out using two antibodies and seven DNA loci, the performance of BioVyon Protein A was significantly better, with a greater percentage of DNA pull-down in all of the assays tested compared with bead-based matrices, Protein A Sepharose, and Dynabeads Protein A. Furthermore, the rigid porous disc format within a column made the BioVyon matrix much easier to use with fewer steps and less equipment requirements, resulting in a significant reduction in the time taken to process the ChIP samples. In summary, BioVyon Protein A provides a column-based assay method for ChIP and other immunoprecipitation-based procedures; the rigid porous structure of BioVyon enables a fast and robust protocol with higher ChIP enrichment ratios.     

Transient excretion of succinate from <Emphasis Type="Italic">Trichoderma atroviride</Emphasis> submerged mycelia reveals the complex movements and metabolism of carboxylates

Submerged growth of Trichoderma atroviride CCM F 534 on glucose-containing medium was accompanied by the excretion of organic acids (succinate, citrate, alpha-ketoglutarate, fumarate, aconitate). The excretion of succinate was transient. After 48–72 h cultivation, millimolar amounts of succinate disappeared from the medium. We studied the mechanism of the removal of succinate from the medium and demonstrated the activation of the inward transport of succinate by submerged mycelia. This transport was carrier-mediated, had a low solute specificity, and was driven by proton-motive force. The last aspect was provided by the activation of the H⁺-ATPase, as documented by measurements of ATPase activity and expression of the pma gene. The disruption of the pma gene abolished the capacity of the mycelia to re-uptake succinate but not its production. Results show that excreted carboxylates could serve as alternative nutrients in the late phase(s) of submerged growth, explain why inward transport system(s) for carboxylates are induced, and indicate that the inward-directed transport could interfere with the production of carboxylic acids by fungi.     

JAK/STAT and the GATA factor Pannier control hemocyte maturation and differentiation in Drosophila

The lymph gland is the major site of hematopoiesis in Drosophila. During late larval stages three types of hemocytes are produced, plasmatocytes, crystal cells, and lamellocytes, and their differentiation is tightly controlled by conserved factors and signaling pathways. JAK/STAT is one of these pathways which have essential roles in vertebrate and fly hematopoiesis. We show that Stat has opposing cell-autonomous and non-autonomous functions in hemocyte differentiation. Using a clonal approach we established that loss of Stat in a set of prohemocytes in the cortical zone induces plasmatocyte maturation in adjacent hemocytes. Hemocytes lacking Stat fail to differentiate into plasmatocytes, indicating that Stat positively and cell-autonomously controls plasmatocyte differentiation. We also identified the GATA factor pannier (pnr) as a downstream target of Stat. By analyzing the phenotypes resulting from clonal loss and over-expression of pnr in lymph glands, we find that Pnr is positively regulated by Stat and specifically required for the differentiation of plasmatocytes. Stat and Pnr represent two essential factors controlling blood cell maturation in the developing lymph gland and exert their functions both in a cell-autonomous and non-cell-autonomous manner.     

Lung surfactant protein SP-B promotes formation of bilayer reservoirs from monolayer and lipid transfer between the interface and subphase

We investigated the possible role of SP-B proteins in the function of lung surfactant. To this end, lipid monolayers at the air/water interface, bilayers in water, and transformations between them in the presence of SP-B were simulated. The proteins attached bilayers to monolayers, providing close proximity of the reservoirs with the interface. In the attached aggregates, SP-B mediated establishment of the lipid-lined connection similar to the hemifusion stalk. Via this connection, a lipid flow was initiated between the monolayer at the interface and the bilayer in water in a surface-tension-dependent manner. On interface expansion, the flow of lipids to the monolayer restored the surface tension to the equilibrium spreading value. SP-B induced formation of bilayer folds from the monolayer at positive surface tensions below the equilibrium. In the absence of proteins, lipid monolayers were stable at these conditions. Fold nucleation was initiated by SP-B from the liquid-expanded monolayer phase by local bending, and the proteins lined the curved perimeter of the growing fold. No effect on the liquid-condensed phase was observed. Covalently linked dimers resulted in faster kinetics for monolayer folding. The simulation results are in line with existing hypotheses on SP-B activity in lung surfactant and explain its molecular mechanism.     

Conservation of a packaging signal and the viral genome RNA packaging mechanism in alphavirus evolution

Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. In infected cells, alphaviruses display remarkable specificity in RNA packaging by encapsidating only their genomic RNA while avoiding packaging of the more abundant viral subgenomic (SG), cellular messenger and transfer RNAs into released virions. In this work, we demonstrate that in spite of evolution in geographically isolated areas and accumulation of considerable diversity in the nonstructural and structural genes, many alphaviruses belonging to different serocomplexes harbor RNA packaging signals (PSs) which contain the same structural and functional elements. Their characteristic features are as follows. (i) Sindbis, eastern, western, and Venezuelan equine encephalitis and most likely many other alphaviruses, except those belonging to the Semliki Forest virus (SFV) clade, have PSs which can be recognized by the capsid proteins of heterologous alphaviruses. (ii) The PS consists of 4 to 6 stem-loop RNA structures bearing conserved GGG sequences located at the base of the loop. These short motifs are integral elements of the PS and can function even in the artificially designed PS. (iii) Mutagenesis of the entire PS or simply the GGG sequences has strong negative effects on viral genome packaging and leads to release of viral particles containing mostly SG RNAs. (iv) Packaging of RNA appears to be determined to some extent by the number of GGG-containing stem-loops, and more than one stem-loop is required for efficient RNA encapsidation. (v) Viruses of the SFV clade are the exception to the general rule. They contain PSs in the nsP2 gene, but their capsid protein retains the ability to use the nsP1-specific PS of other alphaviruses. These new discoveries regarding alphavirus PS structure and function provide an opportunity for the development of virus variants, which are irreversibly attenuated in terms of production of infectious virus but release high levels of genome-free virions.     

Auxin minimum defines a developmental window for lateral root initiation

Root system architecture depends on lateral root (LR) initiation that takes place in a relatively narrow developmental window (DW). Here, we analyzed the role of auxin gradients established along the parent root in defining this DW for LR initiation. Correlations between auxin distribution and response, and spatiotemporal control of LR initiation were analyzed in Arabidopsis thaliana and tomato (Solanum lycopersicum). In both Arabidopsis and tomato roots, a well defined zone, where auxin content and response are minimal, demarcates the position of a DW for founder cell specification and LR initiation. We show that in the zone of auxin minimum pericycle cells have highest probability to become founder cells and that auxin perception via the TIR1/AFB pathway, and polar auxin transport, are essential for the establishment of this zone. Altogether, this study reveals that the same morphogen-like molecule, auxin, can act simultaneously as a morphogenetic trigger of LR founder cell identity and as a gradient-dependent signal defining positioning of the founder cell specification. This auxin minimum zone might represent an important control mechanism ensuring the LR initiation steadiness and the acropetal LR initiation pattern.     

Leaf dehydroascorbate reductase and catalase activity is associated with soil drought tolerance in bread wheat

A number of morphological, physiological and phenological traits have been suggested as significant markers of adaptation to drought in bread wheat (Triticum aestivum L.). This study was aimed at the identification of a relationship between dehydroascorbate reductase (DHAR, EC 1.8.5.1) and catalase (CAT, EC 1.11.1.6) activities in leaves of wheat plants and stability of yield components under water deficit. The single chromosome substitution lines of cv. Chinese Spring carrying separate chromosomes from the donor Synthetic 6x, an artificial hexaploid combining the genomes of the two wild species, Triticum dicoccoides (AABB) and Aegilops tauschii (DD), were the objects of the investigations. The activities of the DHAR and CAT were correlated with flag leaf relative water content and two indexes of stability of grain yield components under drought across the set substitution lines. The lines carrying a synthetic hexaploid homologous pair of chromosomes 1B, 1D, 2D, 3D or 4D all expressed a low constitutive level of DHAR and the lines carrying chromosomes 3B, 1D, 2D and 3D a low constitutive level of CAT. All were able to increase this level (by fourfold for DHAR and by 1.5-fold for CAT) in response to stress caused by water deficit. When challenged by drought stress, these lines tended to be the most effective in retaining the water status of the leaves and preventing the grain yield components from being compromised. The discovered genetic variability for enzymes activity in leaves of wheat might be a useful selection criterion for drought tolerance.     

13,16-Dimethyl octacosanedioic acid (iso-diabolic acid), a common membrane-spanning lipid of Acidobacteria subdivisions 1 and 3

The distribution of membrane lipids of 17 different strains representing 13 species of subdivisions 1 and 3 of the phylum Acidobacteria, a highly diverse phylum of the Bacteria, were examined by hydrolysis and gas chromatography-mass spectrometry (MS) and by high-performance liquid chromatography-MS of intact polar lipids. Upon both acid and base hydrolyses of total cell material, the uncommon membrane-spanning lipid 13,16-dimethyl octacosanedioic acid (iso-diabolic acid) was released in substantial amounts (22 to 43% of the total fatty acids) from all of the acidobacteria studied. This lipid has previously been encountered only in thermophilic Thermoanaerobacter species but bears a structural resemblance to the alkyl chains of bacterial glycerol dialkyl glycerol tetraethers (GDGTs) that occur ubiquitously in peat and soil and are suspected to be produced by acidobacteria. As reported previously, most species also contained iso-C(15) and C(16:1ω7C) as major fatty acids but the presence of iso-diabolic acid was unnoticed in previous studies, most probably because the complex lipid that contained this moiety was not extractable from the cells; it could only be released by hydrolysis. Direct analysis of intact polar lipids in the Bligh-Dyer extract of three acidobacterial strains, indeed, did not reveal any membrane-spanning lipids containing iso-diabolic acid. In 3 of the 17 strains, ether-bound iso-diabolic acid was detected after hydrolysis of the cells, including one branched GDGT containing iso-diabolic acid-derived alkyl chains. Since the GDGT distribution in soils is much more complex, branched GDGTs in soil likely also originate from other (acido)bacteria capable of biosynthesizing these components.     

In vitro mitochondrial effects of PK 11195, a synthetic translocator protein 18 kDa (TSPO) ligand,in human osteoblast-like cells

The role of the TSPO in metabolism of human osteoblasts is unknown. We hypothesized that human osteoblast metabolism may be modulated by the TSPO. Therefore we evaluated the presence of TSPO in human osteoblast-like cells and the effect of its synthetic ligand PK 11195 on these cells. The presence of TSPO was determined by [³H]PK 11195 binding using Scatchard analysis: Bmax 7682 fmol/mg, Kd 9.24 nM. PK 11195 did not affect significantly cell proliferation, cell death, cellular viability, maturation, [¹⁸F]-FDG incorporation and hexokinase 2 gene expression or protein levels. PK 11195 exerted a suppressive effect on VDAC1 and caused an increase in TSPO gene expression or protein levels. In parallel there was an increase in mitochondrial mass, mitochondrial ATP content and a reduction in ΔΨm collapse. Thus, it appears that PK11195 (10⁻⁵ M) stimulates mitochondrial activity in human osteoblast-like cells without affecting glycolytic activity and cell death.     

IL-6 promotes cardiac graft rejection mediated by CD4+ cells

IL-6 mediates numerous immunologic effects relevant to transplant rejection; however, its specific contributions to these processes are not fully understood. To this end, we neutralized IL-6 in settings of acute cardiac allograft rejection associated with either CD8(+) or CD4(+) cell-dominant responses. In a setting of CD8(+) cell-dominant graft rejection, IL-6 neutralization delayed the onset of acute rejection while decreasing graft infiltrate and inverting anti-graft Th1/Th2 priming dominance in recipients. IL-6 neutralization markedly prolonged graft survival in the setting of CD4(+) cell-mediated acute rejection and was associated with decreased graft infiltrate, altered Th1 responses, and reduced serum alloantibody. Furthermore, in CD4(+) cell-dominated rejection, IL-6 neutralization was effective when anti-IL-6 administration was delayed by as many as 6 d posttransplant. Finally, IL-6-deficient graft recipients were protected from CD4(+) cell-dominant responses, suggesting that IL-6 production by graft recipients, rather than grafts, is necessary for this type of rejection. Collectively, these observations define IL-6 as a critical promoter of graft infiltration and a shaper of T cell lineage development in cardiac graft rejection. In light of these findings, the utility of therapeutics targeting IL-6 should be considered for preventing cardiac allograft rejection.