Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation

Docosahexaenoic acid (DHA, 22:6) is an n−3 polyunsaturated fatty acid (n−3 PUFA) that influences immunological, metabolic, and neurological responses through complex mechanisms. One structural mechanism by which DHA exerts its biological effects is through its ability to modify the physical organization of plasma membrane signaling assemblies known as sphingomyelin/cholesterol (SM/chol)-enriched lipid rafts. Here we studied how DHA acyl chains esterified in the sn-2 position of phosphatidylcholine (PC) regulate the formation of raft and non-raft domains in mixtures with SM and chol on differing size scales. Coarse grained molecular dynamics simulations showed that 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) enhances segregation into domains more than the monounsaturated control, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). Solid state ²H NMR and neutron scattering experiments provided direct experimental evidence that substituting PDPC for POPC increases the size of raft-like domains on the nanoscale. Confocal imaging of giant unilamellar vesicles with a non-raft fluorescent probe revealed that POPC had no influence on phase separation in the presence of SM/chol whereas PDPC drove strong domain segregation. Finally, monolayer compression studies suggest that PDPC increases lipid-lipid immiscibility in the presence of SM/chol compared to POPC. Collectively, the data across model systems provide compelling support for the emerging model that DHA acyl chains of PC lipids tune the size of lipid rafts, which has potential implications for signaling networks that rely on the compartmentalization of proteins within and outside of rafts.     

Effects of Lipid Composition and Phase on the Membrane Interaction of the Prion Peptide 106-126 Amide

Lipid rafts are specialized liquid-ordered (L_o) phases of the cell membrane that are enriched in sphingolipids and cholesterol (Chl), and surrounded by a liquid-disordered (L_d) phase enriched in glycerophospholipids. Lipid rafts are involved in the generation of pathological forms of proteins that are associated with neurodegenerative diseases. To investigate the effects of lipid composition and phase on the generation of pathological forms of proteins, we constructed an L_d-gel phase-separated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sphingomyelin (from bovine brain (BSM))-supported lipid bilayer (SLB) and an L_d-L_o phase-separated POPC/BSM/Chl SLB. We used in situ time-lapse atomic force microscopy to study the interactions between these SLBs and the prion peptide K¹⁰⁶TNMKHMAGAAAAGAVVGGLG¹²⁶ (PrP106-126) amide, numbered according to the human prion-peptide sequence. Our results show that: 1), with the presence of BSM in the L_d phase, the PrP106-126 amide induces fully penetrated porations in the L_d phase of POPC/BSM SLB and POPC/BSM/Chl SLB; 2), with the presence of both BSM and Chl in the L_d phase, the PrP106-126 amide induces the disintegration of the L_d phase of POPC/BSM/Chl SLB; and 3), with the presence of both BSM and Chl in the L_o phase, PrP106-126 amide induces membrane thinning in the L_o phase of POPC/BSM/Chl SLB. These results provide comprehensive insight into the process by which the PrP106-126 amide interacts with lipid membranes.     

Temperature induced lipid membrane restructuring and changes in nanomechanics

The naturally occurring milk sphingomyelin is of particular interest owing to its complex composition and involvement in the formation of the milk fat globule membrane (MFGM). Knowledge of membrane organization and nanomechanical stability has proved to be crucial in understanding their properties and functions. In this work, two model membrane systems composed of 1, 2 dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (egg-SM) and cholesterol, and DOPC, milk sphingomyelin (milk-SM) and cholesterol were exposed to both RT and 10 °C. The morphological and nanomechanical changes were investigated using atomic force microscopy (AFM) imaging and force mapping below RT using a designed liquid cell with temperature-control. In both systems, the size and shape of SM/Chol-enriched liquid ordered domains (L_o) and DOPC-enriched liquid disordered phase (L_d) were monitored at controlled temperatures. AFM based force-mapping showed that rupture forces were consistently higher for L_o domains than L_d phases and were decreased for L_d with decreasing temperature while an increase in breakthrough force was observed in L_o domains. More interestingly, dynamic changes and defect formations in the hydrated lipid bilayers were mostly detected at low temperature, suggesting a rearrangement of lipid molecules to relieve additional tension introduced upon cooling. Noteworthy, in these model membrane systems, tension-driven defects generally heal on reheating the sample. The results of this work bring new insights to low temperature induced membrane structural reorganization and mechanical stability changes which will bring us one step closer to understand more complex systems such as the MFGM.     

Direct visualization of the lateral structure of giant vesicles composed of pseudo-binary mixtures of sulfatide,asialo-GM1 and GM1 with POPC

We compared the lateral structure of giant unilamellar vesicles (GUVs) composed of three pseudo binary mixtures of different glycosphingolipid (GSL), i.e. sulfatide, asialo-GM1 or GM1, with POPC. These sphingolipids possess similar hydrophobic residues but differ in the size and charge of their polar head group. Fluorescence microscopy experiments using LAURDAN and DiIC₁₈ show coexistence of micron sized domains in a molar fraction range that depends on the nature of the GSLs. In all cases, experiments with LAURDAN show that the membrane lateral structure resembles the coexistence of solid ordered and liquid disordered phases. Notably, the overall extent of hydration measured by LAURDAN between the solid ordered and liquid disordered membrane regions show marked similarities and are independent of the size of the GSL polar head group. In addition, the maximum amount of GSL incorporated in the POPC bilayer exhibits a strong dependence on the size of the GSL polar head group following the order sulfatide > asialo-GM1 > GM1. This observation is in full harmony with previous experiments and theoretical predictions for mixtures of these GSL with glycerophospholipids. Finally, compared with previous results reported in GUVs composed of mixtures of POPC with the sphingolipids cerebroside and ceramide, we observed distinctive curvature effects at particular molar fraction regimes in the different mixtures. This suggests a pronounced effect of these GSL on the spontaneous curvature of the bilayer. This observation may be relevant in a biological context, particularly in connection with the highly curved structures found in neural cells.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s_o) and liquid-disordered (l_d) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l_o) and l_d phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s_o-l_d and l_o-l_d phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Lo/Ld phase coexistence modulation induced by GM1

Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting “raft-like” L_o domain size and the search for L_o nanodomains as precursors in L_o microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the L_o/L_d lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1 mol % abolishes the formation of the micrometer-scale L_o domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a L_o/L_d lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C₁₂NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale L_o phase domains over an L_d phase is determined, and is shifted to higher values when the GM1 content increases. A “morphological” phase diagram could be made, and it displays three regions corresponding respectively to L_o/L_d micrometric phase separation, L_o/L_d nanometric phase separation, and a homogeneous L_d phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, “arresting” domain growth and thereby stabilizing L_o nanodomains.     

Determination of hippuric acid in human urine by ion chromatography with conductivity detection

A simple, rapid, precise and eco-friendly ion chromatography (IC) method for the determination of hippuric acid (HA) in human urine was proposed in this paper. The separation was carried out an anion exchange column with 2.0 mmol L^?1 Na₂CO₃ + 2.0 mmol L^?1 NaHCO₃ as mobile phase at the flow-rate 0.7 mL min^?1. A suppressed conductivity detector was used and the detection limit was 1.0 μg L^?1(S/N = 3) for hippuric acid. The analysis time for one run was 30 min under the optimized IC condition. The recovery of hippuric acid was 93.2–98.0% while the relative standard deviation (RSD) was 1.4–2.3% by seven measurements.     

Phase behaviour of oleanolic acid,pure and mixed with stearic acid: Interactions and crystallinity

The phase behaviour of pure oleanolic acid (OLA) and in mixtures with stearic acid (SA) was characterized by differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and nuclear magnetic resonance (NMR). The crystalline OLA as received (OLA_ar) becomes amorphous after being dissolved in chloroform and vacuum-dried at 50 °C (OLA₅₀). Upon heating, both forms transform to the needle shape crystalline form (OLA₂₂₀). Dimerization through H-bonding between COOH groups was detected both in OLA_ar and OLA₂₂₀. Dimers are stronger in OLA₂₂₀, where H-bonding also involves the alcohol groups and plays a role in the crystalline organization. A eutectic type phase diagram was established for mixtures, with the eutectic composition close to pure SA. Mixtures rich in SA are miscible in the liquid and in the amorphous solid states, where the presence of SA–OLA co-dimers, formed through H-bonding between carboxyl groups, was detected. Miscibility and SA crystallinity decrease drastically with the OLA content.     

Stable isotope liquid chromatography-tandem mass spectrometry assay for fatty acid amide hydrolase activity

Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the hydrolysis of the endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) to arachidonic acid (AA) and ethanolamine (EA). Published FAAH activity assays mostly employ radiolabeled anandamide or synthetic fluorogenic substrates. We report a stable isotope liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for specific, sensitive, and high-throughput capable FAAH activity measurements. The assay uses AEA labeled with deuterium on the EA moiety (d₄-AEA) as substrate and measures the specific reaction product tetradeutero-EA (d₄-EA) and the internal standard ¹³C₂-EA. Selected reaction monitoring of m/z 66 → m/z 48 (d₄-EA) and m/z 64 → m/z 46 (¹³C₂-EA) in the positive electrospray ionization mode after liquid chromatographic separation on a HILIC (hydrophilic interaction liquid chromatography) column is performed. The assay was developed and thoroughly validated using recombinant human FAAH (rhFAAH) and then was applied to human blood and dog liver samples. rhFAAH-catalyzed d₄-AEA hydrolysis obeyed Michaelis–Menten kinetics (K_M = 12.3 μM, V_max = 27.6 nmol/min mg). Oleoyl oxazolopyridine (oloxa) was a potent, partial noncompetitive inhibitor of rhFAAH (IC₅₀ = 24.3 nM). Substrate specificity of other fatty acid ethanolamides decreased with decreasing length, number of double bonds, and lipophilicity of the fatty acid skeleton. In human whole blood, we detected FAAH activity that was inhibited by oloxa.     

The DNA–DNA spacing in gemini surfactants–DOPE–DNA complexes

Gemini surfactants from the homologous series of alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, number of spacer carbons n = 2 – 12) and dioleoylphosphatidylethanolamine (DOPE) were used for cationic liposome (CL) preparation. CLs condense highly polymerized DNA creating complexes. Small-angle X-ray diffraction identified them as condensed lamellar phase L_α^C in the studied range of molar ratios CnGS12/DOPE in the temperature range 20 – 60 °C. The DNA–DNA distance (d_DNA) is studied in dependence to CnGS12 spacer length and membrane surface charge density. The high membrane surface charge densities (CnGS12/DOPE = 0.35 and 0.4 mol/mol) lead to the linear dependence of d_DNA vs. n correlating with the interfacial area of the CnGS12 molecule.     

Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy

The organization of the plasma membrane is regulated by the dynamic equilibrium between the liquid ordered (L_o) and liquid disordered (L_d) phases. The abundance of the L_o phase is assumed to be a consequence of the interaction between cholesterol and the other lipids, which are otherwise in either the L_d or gel (S_o) phase. The characteristic lipid packing in these phases results in significant differences in their respective lateral dynamics. In this study, imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is applied to monitor the diffusion within supported lipid bilayers (SLBs) as functions of temperature and composition. We show that the temperature dependence of membrane lateral diffusion, which is parameterized by the Arrhenius activation energy (E_Arr), can resolve the sub-resolution phase behavior of lipid mixtures. The FCS diffusion law, a novel membrane heterogeneity ruler implemented in ITIR-FCS, is applied to show that the domains in the S_o–L_d phase are static and large while they are small and dynamic in the L_o–L_d phase. Diffusion measurements and the subsequent FCS diffusion law analyses at different temperatures show that the modulation in membrane dynamics at high temperature (313 K) is a cumulative effect of domain melting and rigidity relaxation. Finally, we extend these studies to the plasma membranes of commonly used neuroblastoma, HeLa and fibroblast cells. The temperature dependence of membrane dynamics for neuroblastoma cells is significantly different from that of HeLa or fibroblast cells as the different cell types exhibit a high level of compositional heterogeneity.     

Inhibition of lecithin:cholesterol acyltransferase activity by synthetic phosphatidylcholine species containing eicosapentaenoic acid or docosahexaenoic acid in the sn-2 position.

Phospholipids isolated from the plasma of monkeys fed a diet enriched in fish oil were poor substrates for cholesteryl ester (CE) synthesis by the lecithin:cholesterol acyltransferase (LCAT) reaction relative to those from animals fed a lard containing diet when the phospholipids were used for the preparation of recombinant particles by cholate dialysis (Parks, J. S., B. C. Bullock, and L. L. Rudel. 1989. J. Biol. Chem. 264: 2545-2551). The purpose of the present study was to directly test the influence of eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3) in the sn-2 position of phosphatidylcholine (PC) on the activity of LCAT. PC species containing 1-palmitoyl-2-oleoyl PC (POPC), 1-palmitoyl-2-linoleoyl PC (PLPC), 1-palmitoyl-2-arachidonoyl PC (PAPC), 1-palmitoyl-2-eicosapentaenoyl PC (PEPC), or 1-palmitoyl-2-docosahexaenoyl PC (PDPC) were purchased or synthesized and made into recombinant particles of uniform size and composition with [14C]cholesterol and apoA-I using the cholate dialysis procedure. The recombinant particles (PC:cholesterol:apoA-I molar ratio = 42:1.9:1) exhibited the following order of reactivity towards purified human LCAT in vitro: POPC greater than PLPC greater than PEPC = PAPC greater than PDPC. The apparent Vmax/Km for recombinant particles containing PEPC and PDPC was 17% and 7% that of particles containing POPC, respectively. There was a linear decrease in CE formation when the percentage of PEPC or PDPC was increased from 0 to 100% relative to POPC in recombinant particles with a constant PC:cholesterol:apoA-I molar ratio, suggesting that the PEPC and PDPC were competitive inhibitors of the LCAT reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple thermodynamic model of the liquid-ordered state and the interactions between phospholipids and cholesterol

A theoretical model is proposed to describe the heat capacity function and the phase behavior of binary mixtures of phospholipids and cholesterol. The central idea is that the liquid-ordered state (L_o) is a thermodynamic state or an ensemble of conformations of the phospholipid, characterized by enthalpy and entropy functions that are intermediate between those of the solid and the liquid-disordered (L_d) states. The values of those thermodynamic functions are such that the L_o state is not appreciably populated in the pure phospholipid, at any temperature, because either the solid or the L_d state have much lower free energies. Cholesterol stabilizes the L_o state by nearest-neighbor interactions, giving rise to the appearance of the L_o phase. The model is studied by Monte Carlo simulations on a lattice with nearest-neighbor interactions, which are derived from experiment as much as possible. The calculated heat capacity function closely resembles that obtained by calorimetry. The phase behavior produced by the model is also in agreement with experimental data. The simulations indicate that separation between solid and L_o phases occurs below the melting temperature of the phospholipid (T_m). Above T_m, small L_d and L_o domains do exist, but there is no phase separation.     

Effect of hydraulic retention time on the treatment of secondary effluent in a subsurface flow constructed wetland

This study evaluates the potential of subsurface flow (SSF) constructed wetlands (CWs) for tertiary treatment of wastewater at four shorter HRTs (1–4 days). The CWs were planted with Typha angustata, which was observed in our earlier study to be more efficient than Phragmites karka and Scirpus littoralis. The CWs comprised four rectangular treatment cells (2.14 m × 0.76 m × 0.61 m) filled with layers of gravel of two different sizes (approximately 2.5 cm and 1.5 cm diameter) to a depth of 0.61 m. The inflow rates of the secondary effluent in the four cells were accordingly fixed at 300 L d^?1, 150 L d^?1, 100 L d^?1 and 75 L d^?1, respectively, for 1, 2, 3 and 4 days HRT. The hydraulic loads ranged between 59.05 mm d^?1 and 236.22 mm d^?1.The wastewater inflow into the CW system as well as the treated effluent were analyzed, using standard methods, at regular intervals for various forms of nitrogen (NH₄-N, NO₃-N and TKN), orthophosphate-P and organic matter (BOD and COD) concentrations over a period of five weeks after the development of a dense stand.The higher HRT of 4 days not only helped maximum removal of all the pollutants but also maintained the stability of the treatment efficiency throughout the monitoring period. For the nutrients (NH₄-N, NO₃-N and TKN), HRT played a more significant role in their removal than in case of organic matter (BOD₃ and COD). More than 90% of NO₃-N and TKN and 100% of NH₄-N were removed from the wastewater at 4 days HRT.At lower HRTs, the mass loading rate was higher with greater fluctuation. However mass reduction efficiency of the T. angustata CW for all forms of nitrogen was >80% with the HRTs of 2, 3 and 4 days.     

Stimuli responsive polymorphism of C12NO/DOPE/DNA complexes: Effect of pH,temperature and composition

N,N-dimethyldodecylamine-N-oxide (C₁₂NO) is a surfactant that may exist either in a neutral or cationic protonated form depending on the pH of aqueous solutions. Using small angle X-ray diffraction (SAXD) we observe the rich structural polymorphism of pH responsive complexes prepared due to DNA interaction with C₁₂NO/dioleoylphosphatidylethanolamine (DOPE) vesicles and discuss it in view of utilizing the surfactant for the gene delivery vector of a pH sensitive system. In neutral solutions, the DNA uptake is low, and a lamellar L_α phase formed by C₁₂NO/DOPE is prevailing in the complexes at 0.2 ≤ C₁₂NO/DOPE < 0.6 mol/mol. A maximum of ~ 30% of the total DNA volume in the sample is bound in a condensed lamellar phase L_α^C at C₁₂NO/DOPE = 1 mol/mol and pH 7.2. In acidic conditions, a condensed inverted hexagonal phase H_II^C was observed at C₁₂NO/DOPE = 0.2 mol/mol. Commensurate lattice parameters, a_HC ≈ d_LC, were detected at 0.3 ≤ C₁₂NO/DOPE ≤ 0.4 mol/mol and pH = 4.9–6.4 suggesting that L_α^C and H_II^C phases were epitaxially related. While at the same composition but pH ~ 7, the mixture forms a cubic phase (Pn3m) when the complexes were heated to 80 °C and cooled down to 20 °C. Finally, a large portion of the surfactant (C₁₂NO/DOPE > 0.5) stabilizes the L_α^C phase in C₁₂NO/DOPE/DNA complexes and the distance between DNA strands (d_DNA) is modulated by the pH value. Both the composition and pH affect the DNA binding in the complexes reaching up to ~ 95% of the DNA total amount at acidic conditions.     

Cultivation of Chlorella vulgaris in tubular photobioreactors: A lipid source for biodiesel production

Chlorella vulgaris was cultivated in two different 2.0 L-helicoidal and horizontal photobioreactors at 5 klux using the bicarbonate contained in the medium and ambient air as the main CO₂ sources. The influence of bicarbonate concentration on biomass growth as well as lipid content and profile was first investigated in shake flasks, where the stationary phase was achieved in about one half the time required by the control. The best NaHCO₃ concentration (0.2 g L⁻¹) was then used in both photobioreactors. While the fed-batch run performed in the helicoidal photobioreactor provided the best result in terms of biomass productivity, which was (84.8 mg L⁻¹ d⁻¹) about 2.5-fold that of the batch run, the horizontal configuration ensured the highest lipid productivity (10.3 mg L⁻¹ d⁻¹) because of a higher lipid content of biomass (22.8%). These preliminary results suggest that the photobioreactor configuration is a key factor either for the growth or the composition of this microalga. The lipid quality of C. vulgaris biomass grown in both photobioreactors is expected to meet the standards for biodiesel, especially in the case of the helicoidal configuration, provided that further efforts will be made to optimize the conditions for its production as a biodiesel source.     

Synthesis and cytotoxicity evaluation of oleanolic acid derivatives

Twelve derivatives of oleanolic acid (1) have been synthesized and evaluated for their inhibitory activities against the growth of prostate PC3, breast MCF-7, lung A549, and gastric BGC-823 cancer cells by MTT assays. Within these series of derivatives, compound 17 exhibited the most potent cytotoxicity against PC3 cell line (IC₅₀ = 0.39 μM) and compound 28 displayed the best activity against A549 cell line (IC₅₀ = 0.22 μM). SAR analysis indicates that H-donor substitution at C-3 position of oleanolic acid may be advantageous for improvement of cytotoxicity against PC3, A549 and MCF-7 cell lines.     

Nitrate removal rates in woodchip media of varying age

A variety of low-cost carbonaceous solids have been successfully tested in bioreactors designed for nitrate treatment. In many agricultural and wastewater settings, however, such reactors may be practical only if they are maintenance free for a number of years after installation. Although field installations have demonstrated consistent treatment over multi-year timeframes, the ability to accurately quantify slowly declining reaction rates in field settings is problematic because of variations in reactor flow rates, ambient temperatures and influent chemistry. In this study, laboratory column tests were undertaken on four samples of coarse wood particle media (woodchips), two that were fresh and two that had been in continuous operation in subsurface denitrifying bioreactors for periods of 2 and 7 years respectively. Four experimental runs were undertaken at increasing influent NO₃-N concentrations of from 3.1 to 48.8 mg N L^?1. Nitrate mass removal rates remained relatively constant and did not systematically increase in successive runs at higher NO₃ concentrations indicating that NO₃ was not the rate-limiting substrate at these concentrations. Thus, zero-order reaction kinetics were used to model the attenuation reaction (presumably denitrification). The 7-year-old media had a mean NO₃-N removal rate of 9.1 mg N L^?1 d^?1 (6.4 g N m^?3 media d^?1), which remained within 75% of the rate for the 2-year-old media (12.1 mg N L^?1 d^?1 or 8.5 g N m^?3 media d^?11) and within 40–59% of the rate for the fresh chips (15.4–23.0 mg N L^?1 d^?1 or 10.8–16.1 g N m^?3 media d^?1). Results support field experience indicating that woodchips loose about 50% of their reactivity during their first year of operation as soluble organic compounds are leached out, but then relatively stable rates persist for a considerable number of years thereafter.     

Nitrate removal and greenhouse gas production in a stream-bed denitrifying bioreactor

Denitrifying bioreactors are currently being tested as an option for treating nitrate (NO₃^?) contamination in groundwater and surface waters. However, a possible side effect of this technology is the production of greenhouse gases (GHG) including nitrous oxide (N₂O) and methane (CH₄). This study examines NO₃^? removal and GHG production in a stream-bed denitrifying bioreactor currently operating in Southern Ontario, Canada. The reactor contains organic carbon material (pine woodchips) intended to promote denitrification. Over a 1 year period, monthly averaged removal of influent (stream water) NO₃^? ranged from 18 to 100% (0.3–2.5 mg N L^?1). Concomitantly, reactor dissolved N₂O and CH₄ production, averaged 6.4 μg N L^?1 (2.4 mg N m^?2 d^?1), and 974 μg C L^?1 (297 mg C m^?2 d^?1) respectively, where production is calculated as the difference between inflow and effluent concentrations. Gas bubbles entrapped in sediments overlying the reactor had a composition ranging from 19 to 64% CH₄, 1 to 6% CO₂, and 0.5 to 2 ppmv N₂O; however, gas bubble emission rates were not quantified in this study. Dissolved N₂O production rates from the bioreactor were similar to emission rates reported for some agricultural croplands (e.g. 0.1–15 mg N m^?2 d^?1) and remained less than the highest rates observed in some N-polluted streams and rivers (e.g. 110 mg N m^?2 d^?1, Grand R., ON). Dissolved N₂O production represented only a small fraction (0.6%) of the observed NO₃^? removal over the monitoring period. Dissolved CH₄ production during summer months (up to 1236 mg C m^?2 d^?1), was higher than reported for some rivers and reservoirs (e.g. 6–66 mg C m^?2 d^?1) but remained lower than rates reported for some wastewater treatment facilities (e.g. sewage treatment plants and constructed wetlands, 19,500–38,000 mg C m^?2 d^?1).     

Kinetics of demetallation of a zinc–salophen complex into liposomes

A Zn–salophen complex has been incorporated into POPC large unilamellar liposomes (LUV) obtained in phosphate buffer at pH 7.4. Fluorescence optical microscopy and anisotropy measurements show that the complex is located at the liposomal surface, close to the polar headgroups. The interaction of the POPC phosphate group with Zn^2 + slowly leads to demetallation of the complex. The process follows first order kinetics and rate constants have been measured fluorimetrically in pure water and in buffered aqueous solution.The coordination of the phosphate group of monomeric POPC with salophen zinc also occurs in chloroform as detected by ESI-MS measurements.The effect of the Zn–salophen complex on the stability of POPC LUV has been evaluated at 25 °C by measuring the rate of release of entrapped 5(6)-carboxyfluorescein (CF) in the presence and in the absence of Triton X-100 as the perturbing agent. It turns out that the inclusion of the complex significantly increases the stability of POPC LUV.