Enzyme-retting of flax and characterization of processed fibers

Enzyme-retting formulations consisting of Viscozyme L, a pectinase-rich commercial enzyme product, and ethylenediaminetetraacetic acid (EDTA) were tested on Ariane fiber flax and North Dakota seed flax straw residue. Flax stems that were crimped to disrupt the outer layers were soaked with various proportions of Viscozyme-EDTA solutions, retted, and then cleaned and cottonized with commercial processing equipment. Fiber properties were determined and crude test yarns were made of raw and Shirley cleaned flax fibers and cotton in various blend levels. Cleaned fibers were obtained from both seed and fiber flax types, but with variations due to treatment. Retting formulations produced fibers having different properties, with enzyme levels of 0.3% (v/v as supplied) giving finer but weaker fibers than 0.05% regardless of EDTA level. Experimental yarns of blended flax and cotton fibers varied in mass coefficient of variation, single end strength, and nep imperfections due to sample and formulation. With cost and fiber and yarn quality as criteria, results established a range in the amounts of components comprising retting formulations as a basis for further studies to optimize enzyme-retting formulations for flax. Under conditions examined herein, Viscozyme L at 0.3% (v/v) plus 25 mM EDTA produced the best test yarns and, therefore, established a base for future studies to develop commercial-grade, short staple flax fibers for use in textiles.     

Characterization of copper interactions with alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42

Cu and Zn have been shown to accumulate in the brains of Alzheimer's disease patients. We have previously reported that Cu(2+) and Zn(2+) bind amyloid beta (Abeta), explaining their enrichment in plaque pathology. Here we detail the stoichiometries and binding affinities of multiple cooperative Cu(2+)-binding sites on synthetic Abeta1-40 and Abeta1-42. We have developed a ligand displacement technique (competitive metal capture analysis) that uses metal-chelator complexes to evaluate metal ion binding to Abeta, a notoriously self-aggregating peptide. This analysis indicated that there is a very-high-affinity Cu(2+)-binding site on Abeta1-42 (log K(app) = 17.2) that mediates peptide precipitation and that the tendency of this peptide to self-aggregate in aqueous solutions is due to the presence of trace Cu(2+) contamination (customarily approximately 0.1 microM). In contrast, Abeta1-40 has much lower affinity for Cu(2+) at this site (estimated log K(app) = 10.3), explaining why this peptide is less self-aggregating. The greater Cu(2+)-binding affinity of Abeta1-42 compared with Abeta1-40 is associated with significantly diminished negative cooperativity. The role of trace metal contamination in inducing Abeta precipitation was confirmed by the demonstration that Abeta peptide (10 microM) remained soluble for 5 days only in the presence of high-affinity Cu(2+)-selective chelators.     

Synthesis of 8-hydroxyquinoline glycoconjugates and preliminary assay of their β1,4-GalT inhibitory and anti-cancer properties

8-Hydroxyquinoline scaffold is a privileged structure used in designing a new active agents with therapeutic potential. Its connections with the sugar unit is formed to improve the pharmacokinetic properties. The broad spectrum of activity of quinoline derivatives, especially glycoconjugates, is often associated with the ability to chelate metal ions or with the ability to intercalate into DNA. Simple and effective methods of synthesis glycoconjugates of 8-hydroxyquinoline and 8-hydroxyquinaldine derivatives, containing an O-glycosidic bond or a 1,2,3-triazole linker in their structure, have been developed. The obtained glycoconjugates were tested for their ability to inhibit β-1,4-Galactosyltransferase, as well as inhibit cancer cell proliferation. It was found that used glycoconjugation strategy influenced both improvement of activity and improvement of the bioavailability of 8-HQ derivatives. Their activity depends on type of attached sugar, presence of protecting groups in sugar moiety and presence of a linker between sugar and quinolone aglycone.     

The Metastasis Suppressor,N-myc Downstream-regulated Gene 1 (NDRG1), Inhibits Stress-induced Autophagy in Cancer Cells

N-myc downstream regulated gene 1 (NDRG1) is a potent metastasis suppressor with an undefined role in the stress response. Autophagy is a pro-survival pathway and can be regulated via the protein kinase-like endoplasmic reticulum kinase (PERK)/eIF2α-mediated endoplasmic reticulum (ER) stress pathway. Hence, we investigated the role of NDRG1 in stress-induced autophagy as a mechanism of inhibiting metastasis via the induction of apoptosis. As thiosemicarbazone chelators induce stress and up-regulate NDRG1 to inhibit metastasis, we studied their effects on the ER stress response and autophagy. This was important to assess, as little is understood regarding the role of the stress induced by iron depletion and its role in autophagy. We observed that the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which forms redox-active iron and copper complexes, effectively induced ER stress as shown by activation of the PERK/eIF2α pathway. Dp44mT also increased the expression of the autophagic marker, LC3-II, and this was dependent on activation of the PERK/eIF2α axis, as silencing PERK prevented LC3-II accumulation. The effect of Dp44mT on LC3-II expression was at least partially due to iron-depletion, as this effect was also demonstrated with the classical iron chelator, desferrioxamine (DFO), and was not observed for the DFO-iron complex. NDRG1 overexpression also inhibited basal autophagic initiation and the ER stress-mediated autophagic pathway via suppression of the PERK/eIF2α axis. Moreover, NDRG1-mediated suppression of the pro-survival autophagic pathway probably plays a role in its anti-metastatic effects by inducing apoptosis. In fact, multiple pro-apoptotic markers were increased, whereas anti-apoptotic Bcl-2 was decreased upon NDRG1 overexpression. This study demonstrates the role of NDRG1 as an autophagic inhibitor that is important for understanding its mechanism of action.     

A comparison of Fe bioavailability and binding of a catecholate siderophore with virus-mediated lysates from the marine bacterium Vibrio alginolyticus PWH3a

We have examined the bioavailability of Fe complexed to a siderophore produced by the heterotrophic marine bacterium Vibrio alginolyticus isolate PWH3a and Fe from ligand-complexes present in virus-mediated lysates (using phage PWH3a-P1) of this same bacterium. Fe-binding functional groups, stability constants and the bioavailability of Fe associated with these two separate ligand pools were determined and contrasted to previous work. Under low-Fe growth conditions, axenic cultures of V. alginolyticus PWH3a were shown to produce catecholate siderophores, while neither catecholate nor hydroxamate-type Fe-binding moieties were detected in virus-generated cell lysates. Analysis of the overall binding strength using electrochemical techniques revealed that the siderophore-containing organic extract and the organics in the virus-mediated lysates had Fe-binding constants comparable to the weaker L₂-type ligands found throughout the water column in seawater. A further purification of the siderophore-containing extract revealed a ligand with a stability constant of logK′_FeL,Fe3+ = 22.3, typical for siderophores and L₁-type of ligands found in marine surface waters. In assimilation studies, the Fe in the lysate was found to be more bioavailable to our model heterotrophic bacterium, autotrophic cyanobacterium and eukaryotic diatom cultures than the catecholate siderophore produced by the Vibrio sp. This work demonstrates that the Fe-containing components of virus-mediated cell lysates are different than siderophores secreted by these same cells, and as such continues to build the argument supporting the importance of virus-mediated Fe regeneration in marine surface waters.     

Chelator-assisted phytoextraction of arsenic,cadmium and lead by Pteris vittata L. and soil microbial community structure response

Abstract

Using biodegradable chelators to assist in phytoextraction may be an effective approach to enhance the heavy-metal remediation efficiencies of plants. A pot experiment was conducted to investigate the effects of ethylenediamine disuccinic acid (EDDS), citric acid (CA), and oxalic acid (OA) on the growth of the arsenic (As) hyperaccumulator Pteris vittata L., its arsenic (As), cadmium (Cd), and lead (Pb) uptake and accumulation, and soil microbial responses in multi-metal(loid)-contaminated soil. The addition of 2.5-mmol kg^?1 OA (OA-2.5) produced 26.7 and 14.9% more rhizoid and shoot biomass, respectively compared with the control, while EDDS and CA treatments significantly inhibited plant growth. The As accumulation in plants after the OA-2.5 treatment increased by 44.2% and the Cd and Pb accumulation in plants after a 1-mmol kg^?1 EDDS treatment increased by 24.5 and 19.6%, respectively. Soil urease enzyme activities in OA-2.5 treatment were significantly greater than those in the control and other chelator treatments (p?<?0.05). A PCR–denatured gradient gel electrophoresis analysis revealed that with the addition of EDDS, CA and OA enhanced soil microbial diversity. It was concluded that the addition of OA-2.5 was suitable for facilitating phytoremediation of soil As and did not have negative effects on the microbial community.     

Antibody-based sensors for heavy metal ions

Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA™ 3000. Four different monoclonal antibodies specific for complexes of EDTA–Cd(II), DTPA–Co(II), 2,9-dicarboxyl-1,10-phenanthroline–U(VI), or cyclohexyl–DTPA–Pb(II) were incubated with the appropriate soluble metal–chelate complex. In the microwell assay format, the immobilized version of the metal–chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10–1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal–chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25±11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81–7.77% and 3.62–14.16% for within run and between run precision, respectively.