Dynamic Control of Electron Transfers in Diflavin Reductases

Diflavin reductases are essential proteins capable of splitting the two-electron flux from reduced pyridine nucleotides to a variety of one electron acceptors. The primary sequence of diflavin reductases shows a conserved domain organization harboring two catalytic domains bound to the FAD and FMN flavins sandwiched by one or several non-catalytic domains. The catalytic domains are analogous to existing globular proteins: the FMN domain is analogous to flavodoxins while the FAD domain resembles ferredoxin reductases. The first structural determination of one member of the diflavin reductases family raised some questions about the architecture of the enzyme during catalysis: both FMN and FAD were in perfect position for interflavin transfers but the steric hindrance of the FAD domain rapidly prompted more complex hypotheses on the possible mechanisms for the electron transfer from FMN to external acceptors. Hypotheses of domain reorganization during catalysis in the context of the different members of this family were given by many groups during the past twenty years. This review will address the recent advances in various structural approaches that have highlighted specific dynamic features of diflavin reductases.     

Non‐natural Olefin Cyclopropanation Catalyzed by Diverse Cytochrome P450s and Other Hemoproteins

Recent work has shown that engineered variants of cytochrome P450_BM3 (CYP102A1) efficiently catalyze non‐natural reactions, including carbene and nitrene transfer reactions. Given the broad substrate range of natural P450 enzymes, we set out to explore if this diversity could be leveraged to generate a broad panel of new catalysts for olefin cyclopropanation (i.e., carbene transfer). Here, we took a step towards this goal by characterizing the carbene transfer activities of four new wild‐type P450s that have different native substrates. All four were active and exhibited a range of product selectivities in the model reaction: cyclopropanation of styrene by using ethyl diazoacetate (EDA). Previous work on P450_BM3 demonstrated that mutation of the axial coordinating cysteine, universally conserved among P450 enzymes, to a serine residue, increased activity for this non‐natural reaction. The equivalent mutation in the selected P450s was found to activate carbene transfer chemistry both in vitro and in vivo. Furthermore, serum albumins complexed with hemin were also found to be efficient in vitro cyclopropanation catalysts.     

Nitric oxide-releasing porous silicon nanoparticles

In this study, the ability of porous silicon nanoparticles (PSi NPs) to entrap and deliver nitric oxide (NO) as an effective antibacterial agent is tested against different Gram-positive and Gram-negative bacteria. NO was entrapped inside PSi NPs functionalized by means of the thermal hydrocarbonization (THC) process. Subsequent reduction of nitrite in the presence of d-glucose led to the production of large NO payloads without reducing the biocompatibility of the PSi NPs with mammalian cells. The resulting PSi NPs demonstrated sustained release of NO and showed remarkable antibacterial efficiency and anti-biofilm-forming properties. These results will set the stage to develop antimicrobial nanoparticle formulations for applications in chronic wound treatment.     

In situ characterization of floc morphology by image analysis in a turbulent Taylor–Couette reactor

Flocculation of bentonite was performed in a turbulent Taylor–Couette reactor under various shear rates. Image processing enabled to determine various morphological characteristics of individual flocs. Not only their mean values but also their distributions were studied under various hydrodynamic conditions. Relevant properties were selected. The temporal evolution of radius of gyration and circularity distributions was monitored during the flocculation process. Although size and shape are obviously correlated, this article points out that their dependency to hydrodynamics is different, showing that flocs of similar sizes produced under different hydrodynamic conditions exhibit different shapes. The sizes are calibrated by the turbulence as the double radius of gyration is close to Kolmogorov microscale, whereas the circularity seems correlated to the rotation speed. © 2014 American Institute of Chemical Engineers AIChE J, 60: 2389–2403, 2014     

Hydrogen storage properties of B‐ and N‐doped microporous carbon

A B‐ and N‐doped microporous carbon has been synthesized via a substitution reaction. The obtained carbon exhibited much higher surface area than the previously reported B‐ and N‐doped carbon. The hydrogen storage measurements indicated that the B‐ and N‐doped microporous carbon had a 53% higher storage capacity than the carbon materials with similar surface areas. Furthermore, hydrogen storage via spillover was studied on Ru‐supported B‐ and N‐doped microporous carbon and a storage capacity of 1.2 wt % at 298 K and 10 MPa was obtained, showing an enhancement factor of 2.2. Ab initio molecular orbital calculations were also performed for the binding energies between the spiltover hydrogen atom and various sites on the doped carbon. The theoretical calculations can explain the experimental results well, which also shed light on the most favorable and possible sites with which the spiltover hydrogen atoms bind. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris

The ability to engineer proteins by directed evolution requiresfunctional expression of the target polypeptide in a recombinanthost suitable for construction and screening libraries of enzymevariants. Bacteria and yeast are preferred, but eukaryotic proteinsoften fail to express in active form in these cells. We haveattempted to resolve this problem by identifying mutations inthe target gene that facilitate its functional expression ina given recombinant host. Here we examined expression of HRPin Saccharomyces cerevisiae. Through three rounds of directedevolution by random point mutagenesis and screening, we obtaineda 40-fold increase in total HRP activity in the S.cerevisiaeculture supernatant compared with wild-type, as measured onABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)](260 units/l/OD₆₀₀). Genes from wild-type and two high-activityclones were expressed in Pichia pastoris, where the total ABTSactivity reached 600 units/l/OD₆₀₀ in shake flasks. The mutantsshow up to 5.4-fold higher specific activity towards ABTS and2.3-fold higher specific activity towards guaiacol.     

Benthic Herbivores are not Deterred by Brevetoxins Produced by the Red Tide Dinoflagellate Karenia Brevis

Gulf of Mexico blooms of the dinoflagellate Karenia brevis produce neurotoxic cyclic polyethers called brevetoxins. During and after a red tide bloom in southwestern Florida, K. brevis cells lyse and release brevetoxins, which then sink to the benthos and coat the surfaces of seagrasses and their epiphytes. We tested the possibility that these brevetoxin-laden foods alter the feeding behavior and fitness of a common benthic herbivore within Floridean seagrass beds, the amphipod Ampithoe longimana. We demonstrated that coating foods with K. brevis extracts that contain brevetoxins at post-bloom concentrations (1 μg g⁻¹ drymass) does not alter the feeding rates of Florida nor North Carolina populations of A. longimana, although a slight deterrent effect was found at eight and ten-fold greater concentrations. During a series of feeding choice assays, A. longimana tended not to be deterred by foods coated with K. brevis extracts nor with the purified brevetoxins PbTx-2 and PbTx-3. Florida juveniles isolated with either extract-coated or control foods for 10 days did not differ in survivorship nor growth. A similar lack of feeding response to brevetoxin-laden foods also was exhibited by two other generalist herbivores of the southeastern United States, the amphipod A. valida and the urchin Arbacia punctulata. Given that benthic mesograzers constitute a significant portion of the diet for the juvenile stage of many nearshore fishes, we hypothesize that the ability of some mesograzers to feed on and retain brevetoxins in their bodies indicates that mesograzers may represent an important route of vertical transmission of brevetoxins through higher trophic levels within Gulf of Mexico estuaries.     

Mechanisms of reorientation and object localization by children: A comparison with rats.

Neurophysiological studies show that the firing of place and head-direction (HD) cells in rats can become anchored to features of the perceptible environment, suggesting that those features partially specify the rat's position and heading. In contrast, behavioral studies suggest that disoriented rats and human children rely exclusively on the shape of their surroundings, ignoring much of the information to which place and HD cells respond. This difference is explored in the current study by investigating young children's ability to locate objects in a square chamber after disorientation. Children 18–24 months old used a distinctive geometric cue but not a distinctively colored wall to locate the object, even after they were familiarized with the colored wall. Results suggest that the spatial representations underlying reorientation and object localization are common to humans and other mammals. Together with the neurophysiological findings, these experiments raise questions for the hypothesis that hippocampal place and HD cells serve as a general orientation device for target localization. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Sesquiterpenoids Lactones: Benefits to Plants and People

Sesquiterpenoids, and specifically sesquiterpene lactones from Asteraceae, may play a highly significant role in human health, both as part of a balanced diet and as pharmaceutical agents, due to their potential for the treatment of cardiovascular disease and cancer. This review highlights the role of sesquiterpene lactones endogenously in the plants that produce them, and explores mechanisms by which they interact in animal and human consumers of these plants. Several mechanisms are proposed for the reduction of inflammation and tumorigenesis at potentially achievable levels in humans. Plants can be classified by their specific array of produced sesquiterpene lactones, showing high levels of translational control. Studies of folk medicines implicate sesquiterpene lactones as the active ingredient in many treatments for other ailments such as diarrhea, burns, influenza, and neurodegradation. In addition to the anti-inflammatory response, sesquiterpene lactones have been found to sensitize tumor cells to conventional drug treatments. This review explores the varied ecological roles of sesquiterpenes in the plant producer, depending upon the plant and the compound. These include allelopathy with other plants, insects, and microbes, thereby causing behavioural or developmental modification to these secondary organisms to the benefit of the sesquiterpenoid producer. Some sesquiterpenoid lactones are antimicrobial, disrupting the cell wall of fungi and invasive bacteria, whereas others protect the plant from environmental stresses that would otherwise cause oxidative damage. Many of the compounds are effective due to their bitter flavor, which has obvious implications for human consumers. The implications of sesquiterpenoid lactone qualities for future crop production are discussed.     

Construction, expression, purification and functional analysis of recombinant NF{kappa}B p50/p65 heterodimer

NFB plays an important role in mediating the gene expressionof numerous cellular processes such as growth, development,the inflammatory response and virus proliferation. The p50/p65heterodimer is the most abundant form of the NFB dimers andplays a more elaborate role in gene regulation. Biochemicalresearch on p50/p65 NFB has not benefited however from the availabilityof easily purified recombinant protein. We report two methodsfor the large scale expression and purification of recombinantNFB p50/p65 heterodimer. The first utilizes a bacterial doubleexpression vector which contains two ribosomal binding sitesto facilitate the coexpression of the polypeptides in the p50/p65NFB heterodimer. The second method uses a mixed protein refoldingstrategy. Both methods yield crystallizable protein. Electrophoreticmobility shift assays confirm that the DNA binding affinityis independent of the method used to purify the protein. Thesemethods will facilitate the numerous studies on various NFB/Relfamily members.