Comparison of organic and inorganic amendments for enhancing soil lead phytoextraction by wheat (Triticum aestivum L.)

Phytoextraction has received increasing attention as a promising, cost-effective alternative to conventional engineering-based remediation methods for metal contaminated soils. In order to enhance the phytoremediative ability of green plants chelating agents are commonly used. Our study aims to evaluate whether, citric acid (CA) or elemental sulfur (S) should be used as an alternative to the ethylene diamine tetraacetic acid (EDTA)for chemically enhanced phytoextraction. Results showed that EDTA was more efficient than CA and S in solubilizing lead (Pb) from the soil. The application of EDTA and S increased the shoot biomass of wheat. However, application of CA at higher rates (30 mmol kg(-1)) resulted in significantly lower wheat biomass. Photosynthesis and transpiration rates increased with EDTA and S application, whereas these parameters were decreased with the application of CA. Elemental sulfur was ineffective for enhancing the concentration of Pb in wheat shoots. Although CA did not increase the Pb solubility measured at the end of experiment, however, it was more effective than EDTA in enhancing the concentration of Pb in the shoots of Triticum aestivum L. It was assumed that increase in Mn concentration to toxic levels in soil with CA addition might have resulted in unusual Pb concentration in wheat plants. The results of the present study suggest that under the conditions used in this experiment, CA at the highest dose was the best amendment for enhanced phytoextraction of Pb using wheat compared to either EDTA or S.     

Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress

Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane-coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.     

Rhomboid cleaves Star to regulate the levels of secreted Spitz

Intracellular trafficking of the precursor of Spitz (Spi), the major Drosophila EGF receptor (EGFR) ligand, is facilitated by the chaperone Star, a type II transmembrane protein. This study identifies a novel mechanism for modulating the activity of Star, thereby influencing the levels of active Spi ligand produced. We demonstrate that Star can efficiently traffic Spi even when present at sub-stoichiometric levels, and that in Drosophila S(2)R(+) cells, Spi is trafficked from the endoplasmic reticulum to the late endosome compartment, also enriched for Rhomboid, an intramembrane protease. Rhomboid, which cleaves the Spi precursor, is now shown to also cleave Star within its transmembrane domain both in cell culture and in flies, expanding the repertoire of known Rhomboid substrates to include both type I and type II transmembrane proteins. Cleavage of Star restricts the amount of Spi that is trafficked, and may explain the exceptional dosage sensitivity of the Star locus in flies.     

Synthesis and performance of 3D-megaporous structures for enzyme immobilization and protein capture

The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross‐linker agent. Ion‐exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation‐induced grafting. The total ionic capacity of such systems was 1.5 mmol H⁺/g, 1.4 mmol H⁺/g, and 17 mmol H⁺/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ?7.2 mg bovine serum albumin/g, ?7.4 hen egg‐white lysozyme (HEWL) mg/g, and ?108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow‐through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D‐megaporous materials described in this work. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Proteomics analysis identifies PARK7 as an important player for renal cell resistance and survival under oxidative stress

Renal fibrosis is a process that is characterized by declining excretory renal function. The molecular mechanisms of fibrosis are not fully understood. Oxidative stress pathways were reported to be involved in renal tissue deterioration and fibrosis progression. In order to identify new molecular targets associated with oxidative stress and renal fibrosis, differential proteomics analysis was performed with established renal cell lines (TK173 and HK-2). The cells were treated with oxidative stress triggering factor H(2)O(2) and the proteome alterations were investigated. Two dimensional protein maps were generated and differentially expressed proteins were processed and identified using mass spectrometry analysis combined with data base search. Interestingly the increase of ROS in the renal cell lines upon H(2)O(2) treatment was accompanied by alteration of a large number of proteins, which could be classified in three categories: the first category grouped the proteins that have been described to be involved in fibrogenesis (e.g. ACTA2, VIN, VIM, DES, KRT, COL1A1, COL4A1), the second category, which was more interesting involved proteins of the oxidative stress pathway (PRDX1, PRDX2, PRDX6, SOD, PARK7, HYOU1), which were highly up-regulated under oxidative stress, and the third category represented proteins, which are involved in different other metabolic pathways. Among the oxidative stress proteins the up-regulation of PARK7 was accompanied by a shift in the pI as a result of oxidation. Knockdown of PARK7 using siRNA led to significant reduction in renal cell viability under oxidative stress. Under H(2)O(2) treatment the PARK7 knockdown cells showed up to 80% decrease in cell viability and an increase in apoptosis compared to the controls. These results highlight for the first time the important role of PARK7 in oxidative stress resistance in renal cells.     

Do physiological roles foster persistence of drug/multidrug-efflux transporters? A case study

Drug and multidrug resistance have greatly compromised the compounds that were once the mainstays of antibiotic therapy. This resistance often persists despite reductions in the use of antibiotics, indicating that the proteins encoded by antibiotic-resistance genes have alternative physiological roles that can foster such persistence in the absence of selective pressure by antibiotics. The recent observations that Tet(L), a tetracycline-efflux transporter, and MdfA, a multidrug-efflux transporter, both confer alkali tolerance offer a striking case study in support of this hypothesis.     

Role of a conserved membrane-embedded acidic residue in the multidrug transporter MdfA

According to the current topology model of the Escherichia coli multidrug transporter MdfA, it contains a membrane-embedded negatively charged residue, Glu26, which was shown to play an important role in substrate recognition. To further elucidate the role of this substrate recognition determinant, various Glu26 replacements were characterized. Surprisingly, studies with neutral MdfA substrates showed that, unlike many enzymatic systems where the size and chemical properties of binding site residues are relatively defined, MdfA tolerates a variety of changes at position 26, including size, hydrophobicity, and charge. Moreover, although efficient transport of positively charged substrates requires a negative charge at position 26 (Glu or Asp), neutralization of this charge does not always abrogate the interaction of MdfA with cationic drugs, thus demonstrating that the negative charge does not play an essential role in the multidrug transport mechanism. Collectively, these results suggest a link between the broad substrate specificity profile of multidrug transporters and the structural and chemical promiscuity at their substrate recognition pockets.