Structural Evidence for the Substrate Channeling of Rice Allene Oxide Cyclase in Biologically Analogous Nazarov Reaction

Allene oxide cyclase (AOC) is a key enzyme in the jasmonic acid (JA) biosynthetic pathway in plants, during which it catalyzes stereospecific conversion of 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid (12,13-EOT) to cis(+)-12-oxophytodienoic acid. Here, rice allene oxide cyclase (OsAOC) was localized to the chloroplast and its native oligomeric structure was analyzed by gel electrophoresis in the absence and presence of a protein-crosslinking reagent. The results suggest that OsAOC exists in solution as a mixture of monomers, dimers, and higher order multimers. OsAOC preferentially exists as dimer at room temperature, but it undergoes temperature-dependent partial denaturation in the presence of SDS. A heteromeric 2:1 complex of OsAOC and rice allene oxide synthase-1 (OsAOS1) was detected after cross-linking. The yield of cis(+)-12-oxophytodienoic acid reached maximal saturation at a 5:1 molar ratio of OsAOC to OsAOS1, when OsAOC and OsAOS1 reactions were coupled. These results suggest that the OsAOC dimer may facilitate its interaction with OsAOS1, and that the heteromeric 2:1 complex may promote efficient channeling of the unstable allene oxide intermediate during catalysis. In addition, conceptual similarities between the reaction catalyzed by AOC and Nazarov cyclization are discussed

Proteomic Profiling and Epitope Analysis of the Complex α-, γ-, and ω-Gliadin Families in a Commercial Bread Wheat

Wheat gliadins are a complex group of proteins that contribute to the functional properties of wheat flour doughs and contain epitopes that are relevant for celiac disease (CD) and wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, we extracted ethanol-soluble gliadin fractions from flour of the Korean bread wheat cultivar Keumkang. Proteins were separated by 2-dimensional gel electrophoresis (2-DE) using a pI range of 6–11 in the first dimension and subjected to tandem mass spectrometry. α-, γ-, and ω-gliadins were identified as the predominant proteins in 31, 28, and one 2-DE spot, respectively. An additional six ω-gliadins were identified in a separate experiment in which a pI range of 3–11 was used for protein separation. We analyzed the composition of CD- and WDEIA-relevant epitopes in the gliadin sequences from Keumkang flour, demonstrating the immunogenic potential of this cultivar. Detailed knowledge about the complement of gliadins accumulated in Keumkang flour provides the background necessary to devise either breeding or biotechnology strategies to improve the functional properties and reduce the adverse health effects of the flour

Immobilization of Soybean Lipoxygenase on Nanoporous Rice Husk Silica by Adsorption: Retention of Enzyme Function and Catalytic Potential

Soybean lipoxygenase was immobilized on nanoporous rice husk silica particles by adsorption, and enzymatic parameters of the immobilized protein, including the efficiency of substrate binding and catalysis, kinetic and operational stability, and the kinetics of thermal inactivation, were investigated. The maximal adsorption efficiency of soybean lipoxygenase to the silica particles was 50%. The desorption kinetics of soybean lipoxygenase from the silica particles indicate that the silica-immobilized enzyme is more stable in an anionic buffer (sodium phosphate, pH 7.2) than in a cationic buffer (Tris-HCl, pH 7.2). The specific activity of immobilized lipoxygenase was 73% of the specific activity of soluble soybean lipoxygenase at a high concentration of substrate. The catalytic efficiency (kcat/Km) and the Michaelis–Menten constant (Km) of immobilized lipoxygenase were 21% and 49% of kcat/Km and Km of soluble soybean lipoxygenase, respectively, at a low concentration of substrate. The immobilized soybean lipoxygenase was relatively stable, as the enzyme specific activity was >90% of the initial activity after four assay cycles. The thermal stability of the immobilized lipoxygenase was higher than the thermal stability of soluble lipoxygenase, demonstrating 70% and 45% of its optimal specific activity, respectively, after incubation for 30 min at 45 °C. These results demonstrate that adsorption on nanoporous rice husk silica is a simple and rapid method for protein immobilization, and that adsorption may be a useful and facile method for the immobilization of many biologically important proteins of interest

Protective effects of combination of Stauntonia hexaphylla and Cornus officinalis on testosterone-induced benign prostatic hyperplasia through inhibition of 5α- reductase type 2 and induced cell apoptosis.

Benign prostatic hyperplasia (BPH) is a progressive pathological condition associated with proliferation of prostatic tissues, prostate enlargement, and lower-urinary tract symptoms. However, the mechanism underlying the pathogenesis of BPH is unclear. The aim of this study was to investigate the protective effects of a combination of Stauntonia hexaphylla and Cornus officinalis (SC extract) on a testosterone propionate (TP)-induced BPH model. The effect of SC extract was examined in a TP-induced human prostate adenocarcinoma cell line. Male Sprague-Dawley rats were randomly divided into 5 groups (n = 6) for in vivo experiments. To induce BPH, all rats, except those in the control group, were administered daily with subcutaneous injections of TP (5 mg/kg) and orally treated with appropriate phosphate buffered saline/drugs (finasteride/saw palmetto/SC extract) for 4 consecutive weeks. SC extract significantly downregulated the androgen receptor (AR), prostate specific antigen (PSA), and 5α-reductase type 2 in TP-induced BPH in vitro. In in vivo experiments, SC extract significantly reduced prostate weight, size, serum testosterone, and dihydrotestosterone (DHT) levels. Histologically, SC extract markedly recovered TP-induced abnormalities and reduced prostatic hyperplasia, thereby improving the histo-architecture of TP-induced BPH rats. SC extract also significantly downregulated AR and PSA expression, as assayed using immunoblotting. Immunostaining revealed that SC extract markedly reduced the 5α-reductase type 2 and significantly downregulated the expression of proliferating cell nuclear antigen. In addition, immunoblotting of B-cell lymphoma 2 (Bcl-2) family proteins indicated that SC extract significantly downregulated anti-apoptotic Bcl-2 and markedly upregulated pro-apoptotic B cell lymphoma-associated X (Bax) expression. Furthermore, SC treatment significantly decreased the Bcl-2/Bax ratio, indicating induced prostate cell apoptosis in TP-induced BPH rats. Thus, our findings demonstrated that SC extract protects against BPH by inhibiting 5α-reductase type 2 and inducing prostate cell apoptosis. Therefore, SC extract might be useful in the clinical treatment of BPH

Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat

Because high-molecular-weight glutenin subunits (HMW-GS) are important contributors to wheat end-use quality, there is a need for high-throughput identification of HMW-GS in wheat genetic resources and breeding lines. We developed an optimized method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to distinguish individual HMW-GS by considering the effects of the alkylating reagent in protein extraction, solvent components, dissolving volume, and matrix II components. Using the optimized method, 18 of 22 HMW-GS were successfully identified in standard wheat cultivars by differences in molecular weights or by their associations with other tightly linked subunits. Interestingly, 1Bx7 subunits were divided into 1Bx7 group 1 and 1Bx7 group 2 proteins with molecular weights of about 82,400 and 83,000 Da, respectively. Cultivars containing the 1Bx7 group 2 proteins were distinguished from those containing 1Bx7OE using well-known DNA markers. HMW-GS 1Ax2* and 1Bx6 and 1By8 and 1By8*, which are difficult to distinguish due to very similar molecular weights, were easily identified using RP-HPLC. To validate the method, HMW-GS from 38 Korean wheat varieties previously evaluated by SDS-PAGE combined with RP-HPLC were analyzed by MALDI-TOF-MS. The optimized MALDI-TOF-MS method will be a rapid, high-throughput tool for selecting lines containing desirable HMW-GS for breeding efforts

Exploiting pi-pi Stacking for Stretchable Semiconducting Polymers

Although intermolecular charge transport is known to occur via pi-pi stacking, the influence of pi-pi stacking on the mechanical properties of polymers has received little attention compared with other dynamic noncovalent interactions. Herein, we demonstrate a method to enhance stretchability via lowering crystallinity and increasing pi-pi stacking of thiophene-based random copolymer chains, which causes pi-pi stacking-induced polymer networks to form within the fully conjugated semiconducting polymer matrix. The polymer networks contain coiled amorphous chains that aid energy dissipation when the polymer film is subjected to strain; furthermore, the pi-pi stacking prevents the chains from irreversible sliding out of place due to the applied strain and provides interchain charge transport. Consequently, we are able to improve the polymer's mechanical properties such as elongation at break, tensile strength, and toughness along with charge mobility. Additionally, our polymer shows great tolerance to a 40% strain without a decrease in mobility while maintaining a stable electrical performance even after 5000 stretching cycles at 30% strain.11Nsciescopu