Participatory plant breeding: a way to arrive at better-adapted onion varieties

The search for varieties that are better adapted to organic farming is a current topic in the organic sector. Breeding programmes specific for organic agriculture should solve this problem. Collaborating with organic farmers in such programmes, particularly in the selection process, can potentially result in varieties better adapted to their needs. Here, we assume that organic farmers' perceptive of plant health is broader than that of conventional breeders. Two organic onion farmers and one conventional onion breeder were monitored in their selection activities in 2004 and 2005 in order to verify whether and in which way this broader view on plant health contributes to improvement of organic varieties. They made selections by positive mass selection in three segregating populations under organic conditions. The monitoring showed that the organic farmers selected in the field for earliness and downy mildew and after storage for bulb characteristics. The conventional breeder selected only after storage. Farmers and breeder applied identical selection directions for bulb traits as a round shape, better hardness and skin firmness. This resulted in smaller bulbs in the breeders’ populations, while the bulbs in the farmer populations were bigger than in the original population. In 2006 and 2007 the new onion populations will be compared with each other and the original populations to determine the selection response

Hybrid Vesicle Stability under Sterilisation and Preservation Processes Used in the Manufacture of Medicinal Formulations

Sterilisation and preservation of vesicle formulations are important considerations for their viable manufacture for industry applications, particular those intended for medicinal use. Here, we undertake an initial investigation of the stability of hybrid lipid-block copolymer vesicles to common sterilisation and preservation processes, with particular interest in how the block copolymer component might tune vesicle stability. We investigate two sizes of polybutadiene-block-poly(ethylene oxide) polymers (PBd12-PEO11 and PBd22-PEO14) mixed with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) considering the encapsulation stability of a fluorescent cargo and the colloidal stability of vesicle size distributions. We find that autoclaving and lyophilisation cause complete loss of encapsulation stability under the conditions studied here. Filtering through 200 nm pores appears to be viable for sterilisation for all vesicle compositions with comparatively low release of encapsulated cargo, even for vesicle size distributions which extend beyond the 200 nm filter pore size. Freeze-thaw of vesicles also shows promise for the preservation of hybrid vesicles with high block copolymer content. We discuss the process stability of hybrid vesicles in terms of the complex mechanical interplay between bending resistance, stretching elasticity and lysis strain of these membranes and propose strategies for future work to further enhance the process stability of these vesicle formulations

Concentrating Membrane Proteins Using Asymmetric Traps and AC Electric Fields

Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol.2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery2006, 5, 993). However, the structure-function relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structure-function determination of these species (Poo et al. Nature1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a “nested trap” and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins

A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H2O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately −240 mV) and low-spin (approximately −110, −190 and −265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (Em=−80 mV) in the presence of NADH (Em=−320 mV) and an NADH–menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.</jats:p

An amicyanin C-terminal loop mutant where the active-site histidine donor cannot be protonated

Protein Chemistry - OU

Membrane mixing and dynamics in hybrid POPC/Poly(1,2-butadiene-block-ethylene oxide) (PBd-b-PEO) lipid/block co-polymer giant vesicles

Lipids and block copolymers can individually self-assemble into vesicles, each with their own particular benefits and limitations. Combining polymers with lipids allows for further optimisation of the vesicle membranes for bionanotechnology applications. Here, POPC lipid is mixed with poly(1,2-butadiene-block-ethylene oxide) of two different molecular weights (PBd22–PEO14, Mr = 1800 g mol−1 and PBd12–PEO11, Mr = 1150 g mol−1) in order to investigate how increasing the polymer fraction affects membrane mixing, hydration and fluidity. Intensity contributions of fluorescently labelled lipid and polymer within mixed GUV membranes confirm membrane homogeneity within the hybrids. General polarisation measurements of Laurdan in GUVs showed little change in membrane hydration as polymer fraction is increased, which suggests good structural compatibility between lipids and polymers that gives rise to well-mixed vesicles. Membrane fluidity in hybrid GUVs was found to decrease non-linearly with increasing polymer fraction. However, the diffusion coefficients for the fluorescent polymer in hybrid membranes did not change significantly with increasing polymer content. While increasing the polymer fraction does reduce the movement of lipids through a polymer-rich matrix, insignificant difference in diffusion coefficients of the polymer suggests that its diffusion is minimally affected by increasing lipid composition in the range studied. These results lay further foundations for the wider development of hybrid vesicles with controlled properties for advanced biotechnologies

Effects of membrane curvature and pH on proton pumping activity of single cytochrome bo3 enzymes

The molecular mechanism of proton pumping by heme-copper oxidases (HCO) has intrigued the scientific community since it was first proposed. We have recently reported a novel technology that enables the continuous characterisation of proton transport activity of a HCO and ubiquinol oxidase from Escherichia coli, cytochrome bo3, for hundreds of seconds on the single enzyme level (Li et al. J Am Chem Soc 137 (2015) 16055–16063). Here, we have extended these studies by additional experiments and analyses of the proton transfer rate as a function of proteoliposome size and pH at the N- and P-side of single HCOs. Proton transport activity of cytochrome bo3 was found to decrease with increased curvature of the membrane. Furthermore, proton uptake at the N-side (proton entrance) was insensitive to pH between pH 6.4–8.4, while proton release at the P-side had an optimum pH of 7.4, suggesting that the pH optimum is related to proton release from the proton exit site. Our previous single-enzyme experiments identified rare, long-lived conformation states of cytochrome bo3 where protons leak back under turn-over conditions. Here, we analyzed and found that 23% of cytochrome bo3 proteoliposomes show ΔpH half-lives below 50 s after stopping turnover, while only 5% of the proteoliposomes containing a non-pumping mutant, E286C cytochrome bo3 exhibit such fast decays. These single-enzyme results confirm our model in which HCO exhibit heterogeneous pumping rates and can adopt rare leak states in which protons are able to rapidly flow back

Detergent-Free Functionalization of Hybrid Vesicles with Membrane Proteins Using SMALPs

Hybrid vesicles (HVs) that consist of mixtures of block copolymers and lipids are robust biomimetics of liposomes, providing a valuable building block in bionanotechnology, catalysis, and synthetic biology. However, functionalization of HVs with membrane proteins remains laborious and expensive, creating a significant current challenge in the field. Here, using a new approach of extraction with styrene-maleic acid (SMA), we show that a membrane protein (cytochrome bo3) directly transfers into HVs with an efficiency of 73.9 ± 13.5% without the requirement of detergent, long incubation times, or mechanical disruption. Direct transfer of membrane proteins using this approach was not possible into liposomes, suggesting that HVs are more amenable than liposomes to membrane protein incorporation from a SMA lipid particle system. Finally, we show that this transfer method is not limited to cytochrome bo3 and can also be performed with complex membrane protein mixtures

The effect of pH and ligand exchange on the redox properties of blue copper proteins

Macromolecular Biochemistr