Role of Se(VI) in counteracting oxidative damage in <Emphasis Type="Italic">Brassica juncea</Emphasis> L. under Cr(VI) stress

Chromium (Cr) is considered to be one of the major environmental hazards and poses a threat to both plant and animal health. Selenium (Se), however, has been recognized as an essential micronutrient in plants. To understand the role of Se(VI) in oxidative stress management and regulation of antioxidative defence mechanism against heavy metal stress, the seedlings of Brassica juncea L. were raised in Petri plates containing nutrient media supplemented with only with Se(VI) and Cr(VI), or their combination. It was observed that of Cr(VI) causes an increase in reactive oxygen species (ROS) in the seedlings leading to oxidative stress. Histological studies using confocal and visible microscopy confirmed the biochemical results. Supplementation of up to 4 µM of Se(VI) to media containing 300 µM of Cr(VI) reduced the contents of ROS and increased enzymatic and non-enzymatic antioxidants in the seedlings. At a concentration of 6 µM, however, Se(VI) was toxic. The results suggested that at appropriate concentrations, the exogenous application of Se(VI) enabled the B. juncea seedlings to counteract the effects of Cr(VI), thereby increasing the resistance of plants.     

Identification and characterization of a spontaneously aggregating amyloid-forming variant of human PrP((90-231)) through phage-display screening of variants randomized between residues 101 and 112

The N-terminal 'unstructured' region of the human prion protein [PrP((90-231))] is believed to play a role in its aggregation because mutations in this region are associated with seeding-independent deposition disorders like Gerstmann-Straussler-Scheinker disease (GSS). One way of examining the effects of such mutations is to search combinatorially derived libraries for sequence variants showing a propensity to aggregate and/or the ability to interact with prion molecules folded into a beta-sheet-based conformation (i.e., beta-PrP or PrP(Sc)). We created a library of 1.8x10(7) variants randomized between positions 101 and 112, displayed it on filamentous bacteriophage, and 'spiked' it with a approximately 25% population of phages-bearing wild-type prion (wt-PrP). Screening was performed through four rounds of biopanning and amplification against immobilized beta-PrP, and yielded three beta-PrP-binding populations: wt-PrP (26% representation) and two non-wt-PrP variants ( approximately 10% and approximately 64% representation, respectively). The remarkable enrichment of one non-wt-PrP variant (MutPrP) incorporating residues KPSKPKTNMKHM in place of KGVLTWFSPLWQ, despite its initial representation at a 5 million-fold lower level than wt-PrP, caused us to produce it and discover: (i) that it readily aggregates into thioflavin-T-binding amyloids between pH 6.0 and 9.0, (ii) that it adopts a soluble beta-sheet based monomeric structure at pH 10.0, (iii) that it is less thermally stable and more compact than wt-PrP, and (iv) that it displays significantly greater resistance to proteolysis than wt-PrP. Our results suggest that sequence variations in the 101-112 region can indeed predispose the prion for aggregation.     

Identification and SAR around N-{2-[4-(2,3-dihydro-benzo[1,4]dioxin-2-ylmethyl)-[1,4]diazepan-1-yl]-ethyl}-2-phenoxy-nicotinamide, a selective alpha2C adrenergic receptor antagonist

The discovery of the CNS-penetrant and selective alpha(2C) adrenergic receptor antagonist N-{2-[4-(2,3-dihydro-benzo[1,4]dioxin-2-ylmethyl)-[1,4]diazepan-1-yl]-ethyl}-2-phenoxy-nicotinamide, 13 is described. Structure-activity studies demonstrate the structural requirements for binding affinity, functional activity, and selectivity over other alpha(2)-AR subtypes.     

1-Toluene-sulfonyl-3-[(3'-hydroxy-5'-substituted)-gamma-butyrolactone]-indoles: synthesis, COX-2 inhibition and anti-cancer activities

Indoles carrying a cyclic ester (gamma-butyrolactone) at C-3 position have been synthesized by the allylation of 3-indoleglyoxylate followed by iodocyclisation and the nucleophilic replacement of the iodo-group. Screening of these molecules for COX-2 inhibition and anti-cancer activities has identified compounds 10 and 11 as highly potent and selective for COX-2 as well as showing remarkable anti-cancer activities (better than that of indomethacin).     

Foldon-guided self-assembly of ultra-stable protein fibers

A common objective in protein engineering is the enhancement of the thermodynamic properties of recombinant proteins for possible applications in nanobiotechnology. The performance of proteins can be improved by the rational design of chimeras that contain structural elements with the desired properties, thus resulting in a more effective exploitation of protein folds designed by nature. In this paper, we report the design and characterization of an ultra-stable self-refolding protein fiber, which rapidly reassembles in solution after denaturation induced by harsh chemical treatment or high temperature. This engineered protein fiber was constructed on the molecular framework of bacteriophage P22 tail needle gp26, by fusing its helical core to the foldon domain of phage T4 fibritin. Using protein engineering, we rationally permuted the foldon upstream and downstream from the gp26 helical core and characterized gp26-foldon chimeras by biophysical analysis. Our data demonstrate that one specific protein chimera containing the foldon immediately downstream from the gp26 helical core, gp26(1-140)-F, displays the highest thermodynamic and structural stability and refolds spontaneously in solution following denaturation. The gp26-foldon chimeric fiber remains stable in 6.0 M guanidine hydrochloride, or at 80 degrees C, rapidly refolds after denaturation, and has both N and C termini accessible for chemical/biological modification, thereby representing an ideal platform for the design of self-assembling nanoblocks.     

Energy and Structure of the M2 Helix in Acetylcholine Receptor-Channel Gating

We studied single-channel currents from neuromuscular acetylcholine receptor-channels with mutations in the pore-lining, M2 helix of the ɛ-subunit. Three parameters were quantified: 1), the diliganded gating equilibrium constant (E₂), which reflects the energy difference between C(losed) and O(pen) conformations; 2), the correlation between the opening rate constant and E₂ on a log-log scale (Φ), which illuminates the energy character of the residue (C- versus O-like) within the C↔O isomerization process; and 3), the open-channel current amplitude (i₀), which reports whether a mutation alters the energetics of ion permeation. The largest E₂ changes were observed in the cytoplasmic half of ɛM2 (5′, 9′, 12′, 13′, and 16′), with smaller changes apparent for residues ≥17′. Φ was ∼0.54 for most ɛM2 residues, but was ∼0.32 at the positions that had largest E₂ changes. An arginine substitution reduced i₀ significantly at six positions, with the magnitude of the reduction increasing, 16′→2′. The measurements suggest that the 9′, 12′, and 13′ residues experience large and late free-energy changes in the channel-opening process. We speculate that in the gating isomerization the pore-facing residues >6′ and <16′ experience multiple energy perturbations associated with changes in protein structure and, perhaps, hydration.     

Survey and phylogenetic analysis of culturable microbes in the oral secretions of three bark beetle species

In a recent study, we reported a previously undescribed behavior in which a bark beetle exuded oral secretions containing bacteria that have antifungal properties, and hence defend their galleries against pervasive antagonistic Hyphomycete fungi. Actinobacteria, a group known for their antibiotic properties, were the most effective against fungi that invade the spruce beetle galleries. In the present study, we describe the isolation and identification of microorganisms from oral secretions of three bark beetles (Coleoptera: Curculionidae: Scolytinae): the spruce beetle, Dendroctonus rufipennis Kirby, the mountain pine beetle, Dendroctonus ponderosae Hopkins, and the pine engraver, Ips pini Say. Bacteria isolated from these three species span the major bacterial classes α-, β-, and γ-Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, except for D. ponderosae , which yielded no α-proteobacteria or Bacteroidetes isolates. Spruce beetles and pine engraver beetles had similar numbers of α-proteobacteria isolates, but pine engravers yielded twice as many Bacteroidetes isolates as spruce beetles. In contrast, mountain pine beetles yielded more isolates in the β- and γ-proteobacteria than spruce beetles and pine engravers. The highest percentage of Actinobacteria was obtained from spruce beetles, followed by pine engravers and mountain pine beetles. All of the fungal isolates obtained from the three beetle species were Ascomycetes. The greatest fungal diversity was obtained in spruce beetles, which had nine species, followed by pine engravers with five, and mountain pine beetles with one.     

Structural elucidation, modification and characterization of seed gum from Cassia javahikai seeds: A non-traditional source of industrial gums

A water-soluble seed gum was isolated from seed endosperm of Cassia javahikai. The acid-catalyzed fragmentation, methylation, selective enzymatic degradation and periodate oxidation suggested a heteropolymeric structure for the polysaccharide. The polysaccharide was shown to have a linear chain of β(1 → 4) linked d-mannopyranosyls units with side chains of α(1 → 6) d-galactopyranosyl units. Grafting of polyacrylamide onto the gum was performed using K₂S₂O₈/ascorbic acid redox system in presence of Ag⁺ as catalyst at 35 ± 2 °C. The viscosity of the gum solution increased on grafting and the grafted gum was observed to resist biodegradation for more than 256 h. Thermogravimetric analysis revealed that grafted gum was more thermally stable than native gum.