PHLOROGLUCINOL DERIVATIVES AND FLAVONES FROM HELICHRYSUM PARONYCHIOIDES

Investigation of Helichrysum paronychioides afforded a total of nine compounds: 4 phloroglucinol derivatives, 2 of which are novel natural products, and 5 flavone derivatives. Structures were established by various spectroscopic techniques (NMR, MS, UV, IR, CD) and by comparison with literature data for the known compounds. The four phloroglucinols, trans-(2R,3R)-5,7-dihydroxy-2,3-dimethyl-4-chromanone (1), 2-butanoyl-4-prenyl-1-methoxy phloroglucinol (2), 2-(2-methylpropanoyl)-4-prenylphloroglucinol (3) and 2-(2-methyl- butanoyl)-4-prenylphloroglucinol (4) were screened for antioxidant activity against Cu-induced LDL oxidation. Compound 4 was found to be the most active inhibiting LDL oxidation at all concentrations (0.5-10 μM) while the other three showed moderate to no activity. KEY WORDS: Helichrysum paronychioides, Asteraceae, Phloroglucinol derivatives, CD spectroscopy, Synthesis Bull. Chem. Soc. Ethiop. 2006, 20(1), 61-68

Data Assimilation Using The Ensemble Kalman Filter With Emphasis On The Inequality Constraints

The reliability of reservoir models generally increases as more data are included during their construction. In the recent past, the ensemble Kalman lter (EnKF) technique has established itself as a viable data assimilation method for models with large numbers of variables. The standard implementation of the EnKF and its variants are being used in a number of disciplines including reservoir engineering.The standard formulation of the EnKF, however, does not take into account the physical constraints on state variables during the data assimilation step so, the constraint violations are often handled heuristically. When the standard implementation of the EnKF is used for history matching, the resulting updates to reservoir propertiessometimes exceed physical bounds, especially when the problem has highly nonlinear model dynamics, the state variables are non-Gaussian, or the ensemble size is small. The physical constraints often contain valuable information about the system which are critical for the valid initialization and the forecast. A successful enforcing of these constraints during the data assimilation step of the EnKF is necessary for valid estimation of reservoir properties.In the conventional EnKF approach, phase saturations (in case of black-oil models) and molar densities (in case of compositional models) are often included in the state vector. Unfortunately, the analysis step of the EnKF sometimes results in non-physical values of phase saturations or molar densities. In this dissertation, we illustrate the problem of using the standard EnKF with a compositional model in which the updated CO2 molar density in some regions of the model space is observed to take negative values while molar densities of the remaining components are increased. A number of solutions to the problem of constraint violation have been proposed in the past including the iterative EnKF, transformation of state variables, reparameterization, and truncation. The standard truncation schemes avoid negative values of molar densities, but do not address the problem of increased molar densities of other components. The results can include a spurious increase in reservoirpressure with a subsequent inability to maintain injection. In this dissertation, we present two dierent methods for incorporating the inequality constraints into the EnKF methodology.In the first part of this dissertation we present a method for the constrained EnKF (CEnKF) which takes into account the physical constraints on the plausible values of state variables during the data assimilation such that the resulting solution is as close as possible to the unconstrained solution obtained from the standard EnKF, and atthe same time, it lies in the feasible region. The proposed method can be implemented in two dierent approaches, both of which convert the inequality constraints to a small number of equality constraints. The first approach uses Lagrange multipliers to enforce the active constraints. In the second approach, the active constraints are used as virtual observations for calibrating the model parameters within plausible ranges. Applying the CEnKF technique in an iterative manner ensures that the resulting solution is within the limits set by the constraints.The application of the proposed CEnKF method is successfully demonstrated on a synthetic 1D linear problem, on a synthetic 2D compositional model, and on a highly heterogeneous three-phaseflow reservoir model. The effect of the constraints on mass conservation is illustrated using a 1D Buckley-Leverett flow example. Results showthat the CEnKF technique is able to enforce the non-negativity constraints on molar densities and the bound constraints on phase saturations (all phase saturations must be between a lower and an upper bound), and achieve a better estimation of reservoir properties than is obtained using only truncation with EnKF.An interior-point method for incorporating the inequality constraints into the EnKF methodology (IPCEnKF) is presented in the second part of this dissertation. In this approach, the objective function for data assimilation is reformulated by adding a barrier function to penalize proximity of the state variables to the boundaries of the feasible region and to set a barrier on the state variables against leaving the feasible region. By doing so, the original constrained optimization problem is transformedinto an unconstrained optimization problem. We present the solution of the resulting unconstrained problem in the form of a new iterative EnKF scheme which implicitly contains inequality constraints on the state variables. The proposed IPCEnKF method is efficient compared to the CEnKF as it does not require the identication of the active constraints. Although the method is iterative to reduce the effect of the barrier term at each assimilation step, the iterations do not require running thesimulator.The IPCEnKF method is successfully tested first on a 1D linear example to illustrate the performance when nonlinearity is not an issue, then on a more realistic 3D, three-phase reservoir flow assimilation problem based on the modied SPE9 model. Results from the reservoir problem show the effectiveness of the newly proposed IPCEnKF method in matching the observations and honoring the inequality constraintson phase saturations. The proposed method is able to achieve a better estimate of reservoir properties than is obtained using only truncation with the standard EnKF

Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli

Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli BE host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity. This allowed preparation of 149 porin mutants in E. coli used in detailed explorations of the structure and function of this membrane protein to high resolutio

Ionic partition and transport in multi-ionic channels: A Molecular Dynamics Simulation study of the OmpF bacterial porin

We performed all-atom molecular dynamics simulations studying the partition of ions and the ionic current through the bacterial porin OmpF and two selected mutants. The study is motivated by new interesting experimental findings concerning their selectivity and conductance behaviour at neutral pH. The mutations considered here are designed to study the effect of removal of negative charges present in the constriction zone of the wild type OmpF channel (which contains on one side a cluster with three positive residues and on the other side two negatively charged residues). Our results show that these mutations induce an exclusion of cations from the constriction zone of the channel, substantially reducing the flow of cations. In fact, the partition of ions inside the mutant channels is strongly inhomogeneous, with regions containing excess of cations and regions containing excess of anions. Interestingly, the overall number of cations inside the channel is larger than the number of anions in the two mutants, as in the OmpF wild type channel. We found that the differences in ionic charge inside these channels are justified by the differences in electric charge between the wild type OmpF and the mutants, following an electroneutral balance

Pseudomonas putida CSV86: A Candidate Genome for Genetic Bioaugmentation

Peer reviewe

Durable vesicles for reconstitution of membrane proteins in biotechnology

The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with

Insights into the function of ion channels by computational electrophysiology simulations

Ion channels are of universal importance for all cell types and play key roles in cellular physiology and pathology. Increased insight into their functional mechanisms is crucial to enable drug design on this important class of membrane proteins, and to enhance our understanding of some of the fundamental features of cells. This review presents the concepts behind the recently developed simulation protocol Computational Electrophysiology (CompEL), which facilitates the atomistic simulation of ion channels in action. In addition, the review provides guidelines for its application in conjunction with the molecular dynamics software package GROMACS. We first lay out the rationale for designing CompEL as a method that models the driving force for ion permeation through channels the way it is established in cells, i.e., by electrochemical ion gradients across the membrane. This is followed by an outline of its implementation and a description of key settings and parameters helpful to users wishing to set up and conduct such simulations. In recent years, key mechanistic and biophysical insights have been obtained by employing the CompEL protocol to address a wide range of questions on ion channels and permeation. We summarize these recent findings on membrane proteins, which span a spectrum from highly ion-selective, narrow channels to wide diffusion pores. Finally we discuss the future potential of CompEL in light of its limitations and strengths. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov

Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current

One major determinant of the efficacy of antibiotics on Gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within the pore, thus partially blocking the ionic current. The modulation of one affinity site by Mg2+ reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg2+-free media are converted to flickery sojourns at the highest apparent current level (all three pores flickering). This indicates weaker binding in the presence of Mg2+. Analysis of the resulting amplitude histograms with beta distributions revealed the rate constants of blocking (k(OB)) and unblocking (k(BO)) in the range of 1,000 to 120,000 s(-1). As expected for a bimolecular reaction, k(OB) was proportional to blocker concentration and k(BO) independent of it. k(OB) was approximately three times lower for enrofloxacin coming from the cis side than from the trans side. The block was not complete, leading to a residual conductivity of the blocked state being similar to 25% of that of the open state. Interpretation of the results has led to the following model: fast flickering as caused by interaction of Mg2+ and enrofloxacin is related to the binding site at the trans side, whereas the cis site mediates slow blocking events which are also found without Mg2+. The difference in the accessibility of the binding sites also explains the dependency of k(OB) on the side of enrofloxacin addition and yields a means of determining the most plausible orientation of OmpF in the bilayer. The voltage dependence suggests that the dipole of the antibiotic has to be adequately oriented to facilitate binding