Temperature dependence of the Langmuir monolayer packing of mycolic acids from Mycobacterium tuberculosis

Phase diagrams of the Langmuir monolayer of dicyclopropyl alpha mycolic acid (α-MA), cyclopropyl methoxy mycolic acid (MeO-MA), and cyclopropyl ketomycolic acids (Keto-MA) from Mycobacterium tuberculosis were obtained by thermodynamic analysis of the surface pressure (π) vs. average molecular area (A) isotherms at temperatures in the range of 10-46 °C. The Langmuir monolayers of MAs were shown to exhibit various phases depending on the temperature (T) and the π values. In the Langmuir monolayer of Keto-MA, the carbonyl group in the meromycolate chain apparently touches the water surface to give the molecule a W-shape in all the temperatures and surface pressures studied. Keto-MA formed a rigid solid condensed film, with four hydrocarbon chains packing together, not observed in the others. In contrast, the monolayer films of α-and MeO-MAs having no such highly hydrophilic intra-chain groups in the meromycolate chain were mostly in liquid condensed phase. This novel insight into the packing of mycolic acids opens up new avenues for the study of the role of mycolic acids in the mycobacterial cell envelopes and pathogenic processes.     

Small-angle X-ray scattering and rheological characterization of alginate gels. 3. Alginic acid gels

Alginic acid gels were studied by small-angle X-ray scattering and rheology to elucidate the influence of alginate chemical composition and molecular weight on the gel elasticity and molecular structure. The alginic acid gels were prepared by homogeneous pH reduction throughout the sample. Three alginates with different chemical composition and sequence, and two to three different molecular weights of each sample were examined. Three alginate samples with fractions of guluronic acid residues of 0.39 (LoG), 0.50 (InG), and 0.68 (HiG), covering the range of commercially available alginates, were employed. The excess scattering intensity I of the alginic acid gels was about 1 order of magnitude larger and exhibited a stronger curvature toward low q compared to ionically cross-linked alginate. The I(q) were decomposed into two components by assuming that the alginic acid gel is composed of aggregated multiple junctions and single chains. Time-resolved experiments showed a large increase in the average size of aggregates and their weight fraction within the first 2 h after onset of gelling, which also coincides with the most pronounced rheological changes. At equilibrium, little or no effect of molecular weight was observed, whereas at comparable molecular weights, an increased scattering intensity with increasing content of guluronic acid residues was recorded, probably because of a larger apparent molecular mass of domains. The results suggest a quasi-ordered junction zone is formed in the initial stage, followed by subsequent assembling of such zones, forming domains in the order of 50 A. The average length of the initial junction zones, being governed by the relative fraction of stabilizing G-blocks and destabilizing alternating (MG) blocks, determines the density of the final random aggregates. Hence, high-G alginates give alginic acid gels of a higher aggregate density compared to domains composed of loosely packed shorter junction zones in InG or LoG system.     

Ionic and acid gel formation of epimerised alginates; the effect of AlgE4

AlgE4 is a mannuronan C5 epimerase converting homopolymeric sequences of mannuronate residues in alginates into mannuronate/guluronate alternating sequences. Treating alginates of different biological origin with AlgE4 resulted in different amounts of alternating sequences. Both ionically cross-linked alginate gels as well as alginic acid gels were prepared from the epimerised alginates. Gelling kinetics and gel equilibrium properties were recorded and compared to results obtained with the original non-epimerised alginates. An observed reduced elasticity of the alginic acid gels following epimerisation by AlgE4 seems to be explained by the generally increased acid solubility of the alternating sequences. Ionically (Ca(2+)) cross-linked gels made from epimerised alginates expressed a higher degree of syneresis compared to the native samples. An increase in the modulus of elasticity was observed in calcium saturated (diffusion set) gels whereas calcium limited, internally set alginate gels showed no change in elasticity. An increase in the sol-gel transitional rate of gels made from epimerised alginates was also observed. These results suggest an increased possibility of creating new junction zones in the epimerised alginate gel due to the increased mobility in the alginate chain segments caused by the less extended alternating sequences.     

FTIR spectroscopy of NH3 on acidic and ionotropic alginate aerogels

The acidity of alginate aerogel films has been investigated by infrared spectroscopy of adsorbed NH(3). Supercritical drying of the alginate provided samples with a surface area of several hundred square meters per gram, in which the probe molecule could reach all acidic sites. Free carboxylic groups were studied on acid-gelled alginates and were found to behave as effective Br?nsted acid sites. Ionotropic alginate gels formed by alkaline earth cations presented only the Lewis acidity of the cations. Ionotropic gels formed by transition metal cations presented both Lewis and Br?nsted sites, because of the presence of a fraction of free carboxylic groups. The incomplete salification was correlated to the pH of the gelling solutions.     

Alginate synthesis by mucoid strains of Pseudomonas aeruginosa PAO

Summary Alginic acid production by Pseudomonas aeruginosa PAO strains was studied in yeast extract/2% (w/v) gluconate medium. In all of the five strains studied, synthesis of the alginic acid was shown to occur in the stationary phase of growth. Each strain produced similar amounts of alginic acid at both 30° C and 37° C. However the amount of alginic acid varied from 7.5–11.5 gl^–1 depending upon the strain. The alginic acid was isolated, purified and its chemical composition determined. All strains produced a polysaccharide rich in polymannuronic acid which contained only polymannuronic acid blocks, polyguluronic acid blocks appeared to be absent. The amount of O-acetylation varied considerably from 2.3–14.7%. Analysis of the chain length distribution by poly-acrylamide gel electropheresis indicated that a homogeneous size of polymer was synthesised when compared to a high viscosity algal sample.     

A comparative study of the phase transitions of phospholipid bilayers and monolayers

Phase transitions in bilayers and monolayers of various synthetic phospholipids with different chain lengths as well as different polar head groups were studied by differential scanning calorimetry or with the film balance technique, respectively. With the film balance, area versus temperature curves (isobars) were recorded at different surface pressures. The monolayer phase transition from the fluid-condensed to the fluid-expanded phase is shifted towards higher temperature when the lateral pressure in the monolayer is increased. The temperature dependence of the equilibrium pressure as well as the magnitude of the area change at the transition depends only on the nature of the phospholipid head group and not on the chain length of the hydrocarbon chains of the lipid. Phospholipids with strong intermolecular attractive interactions between the head groups show low values for dpi/dTm and for the area change, deltaf, whereas phospholipids with negatively charged head groups without intermolecular attractive forces exhibit higher values for dpi/dTm and deltaf. The shift of the monolayer phase transition temperature when increasing the chain length of the lipid is almost identical to the shift in Tm observed for the bilayer system of the same phospholipids. A comparison of monolayer and bilayer systems on the basis of the absolute value of the molecular area of the phospholipid in the bilayer gel phase and the change in area at the bilayer and monolayer transition leads to the following conclusions. The behaviour of the bilayer system is very similar to that of the respective monolayer system at a lateral pressure of approx. 30 dyne/cm, because at this pressure the absolute area and the area change in both systems are the same. Further support for this conclusion comes from the experimental finding that a lateral pressure of 30 dyne/cm the shift in Tm due to the increase in charge when the methyl ester of phosphatidic acid is investigated is the same for the bilayer and the monolayer system.     

Morphology, molecular dynamics and electric conductivity of carbohydrate polymer films based on alginic acid and benzimidazole

The present paper describes a preparation method and molecular investigations of new biodegradable proton-conducting carbohydrate polymer films based on alginic acid and benzimidazole. Electric conductivity was studied in a wide temperature range in order to check the potential application of these compounds as membranes for electrochemical devices. Compared to pure alginic acid powder or its film, the biodegradable film of alginic acid with an addition of benzimidazole exhibits considerably higher conductivity in the range above water boiling temperature (up to approximately 10⁻³ S/cm at 473 K). Due to this important feature the obtained films can be considered as candidates for application in high-temperature electrochemical devices. The microscopic nature and mechanism of the conduction in alginate based materials were studied by proton nuclear magnetic resonance (NMR). The results show specific changes in morphology and molecular dynamics between pure alginate powders and the films obtained without and with the addition of benzimidazole molecules.     

Constant normal pressure, constant surface tension, and constant temperature molecular dynamics simulation of hydrated 1,2-dilignoceroylphosphatidylcholine monolayer

A constant normal pressure, constant surface tension, and constant temperature (NP(N)gammaT) molecular dynamics (MD) simulation of the liquid condensed phase of a 1,2-dilignoceroylphosphatidylcholine (DLGPC) monolayer has been performed at 293.15 K. A DLGPC molecule has two saturated 24-carbon acyl chains, giving the hydrocarbon core thickness of the monolayer approximately 28 A, which is close to the hydrocarbon core thickness of a membrane of a living system. NP(N)gammaT ensemble was used to reproduce the experimental observations, such as area/lipid, because surface tension is an essential factor in determining the monolayer structure. Data analysis on DLGPC/water monolayer shows that various liquid condensed-phase properties of the monolayer have been well reproduced from the simulation, indicating that surface tension 22.9 mN/M used in the simulation is an appropriate condition for the condensed-phase NP(N)gammaT simulation. The simulation results suggest that this long-chain phospholipid monolayer shares many structural characteristics with typical short-chain 1,2-diacylphosphatidylcholine systems, such as DPPC/water monolayer in the condensed phase and DPPC/water bilayer in the gel phase. Furthermore, it was found that DLGPC/water monolayer has almost completely rotationally disordered acyl chains, which have not been observed so far in short-chain 1,2-diacylphosphatidylcholine/water bilayers. This study indicates the good biological relevance of the DLGPC/water monolayer which might be useful in protein/lipid studies to reveal protein structure and protein/lipid interactions in a membrane environment.     

Effect of glycyrrhetinic acid on lipid raft model at the air/water interface

To investigate an interfacial behavior of the aglycon of glycyrrhizin (GC), glycyrrhetinic acid (GA), with a lipid raft model consisting of equimolar ternary mixtures of N-palmitoyl sphingomyelin (PSM), dioleoylphosphatidylcholine (DOPC), and cholesterol (CHOL), Langmuir monolayer techniques were systematically conducted. Surface pressure (π)–molecular area (A) and surface potential (ΔV)–A isotherms showed that the adsorbed GA at the air/water interface was desorbed into the bulk upon compression of the lipid monolayer. In situ morphological analysis by Brewster angle microscopy and fluorescence microscopy revealed that the raft domains became smaller as the concentrations of GA in the subphase (C_GA) increased, suggesting that GA promotes the formation of fluid networks related to various cellular processes via lipid rafts. In addition, ex situ morphological analysis by atomic force microscopy revealed that GA interacts with lipid raft by lying down at the surface. Interestingly, the distinctive striped regions were formed at C_GA = 5.0 μM. This phenomenon was observed to be induced by the interaction of CHOL with adsorbed GA and is involved in the membrane-disrupting activity of saponin and its aglycon. A quantitative comparison of GA with GC (Sakamoto et al., 2013) revealed that GA interacts more strongly with the raft model than GC in the monolayer state. Various biological activities of GA are known to be stronger than those of GC. This fact allows us to hypothesize that differences in the interactions of GA/GC with the model monolayer correlate to their degree of exertion for numerous activities.     

An Analytic Study on the Effect of Alginate on the Velocity Profiles of Blood in Rectangular Microchannels Using Microparticle Image Velocimetry

It is desired to understand the effect of alginic acid sodium salt from brown algae (alginate) as a viscosity modifier on the behavior of blood in vitro using a micro-particle image velocimetry (µPIV) system. The effect of alginate on the shape of the velocity profile, the flow rate and the maximum velocity achieved in rectangular microchannels channels are measured. The channels were constructed of polydimethylsiloxane (PDMS), a biocompatible silicone. Porcine blood cells suspended in saline was used as the working fluid at twenty percent hematocrit (H = 20). While alginate was only found to have minimal effect on the maximum velocity and the flow rate achieved, it was found to significantly affect the shear rate at the wall by between eight to a hundred percent.     

Physical and surface properties of insect apolipophorin III

Apolipophorin III (apoLp-III) from Manduca sexta has a molecular weight of 18,100. Based on its hydrodynamic properties (sedimentation and diffusion coefficients, frictional ratio, intrinsic viscosity) and its behavior during gel permeation chromatography, we concluded that apoLp-III is a prolate ellipsoid with an axial ratio of about 3. The circular dichroic spectrum of apoLp-III suggests that the protein contains approximately 50% alpha-helix. At the air-water interface, apoLp-III forms a monolayer which is gaseous at surface pressures less than or equal to 1 dyne/cm. The isotherm of this phase yields an excluded molecular area of 3800 A2/molecule (23 A2/amino acid). At a surface pressure of 22.1 dynes/cm, the monolayer undergoes a phase transition reminiscent of a first-order phase transition of pure lipids. The monolayer can be compressed in this surface pressure range to an area per molecule of 480 A2 (2.9 A2/amino acid). Since a globular protein of molecular weight 18,100 could occupy an area of only about 2000 A2 when bound to a surface, it is suggested that in the expanded state, apoLp-III must unfold on the surface, whereas in the compressed state, the molecule is oriented with its minor axis parallel to the water surface. ApoLp-III binds with high affinity (Kd = 1.9 X 10(-7)M) to both phosphatidylcholine- and diacylglycerol-coated polystyrene beads. All of these results are consistent with the proposal that apoLp-III plays a key role in increasing the capacity of the insect lipoprotein, lipophorin, to transport diacylglycerol by stabilizing the increment of lipid-water interface that results from diacylglycerol uptake.     

Chemical modification of alginates in organic solvent systems

Alginates are (1→4)-linked linear copolysaccharides composed of β-D-mannuronic acid (M) and its C-5 epimer, α-l-guluronic acid (G). Several strategies to synthesize organically modified alginate derivatives have been reported, but almost all chemistries are performed in either aqueous or aqueous-organic media. The ability to react alginates homogeneously in organic solvents would open up access to a wide range of new chemistries and derivatives. However, past attempts have been restricted by the absence of methods for alginate dissolution in organic media. We therefore report a strategy to dissolve tetrabutylammonium (TBA) salts of alginic acid in polar aprotic solvents containing tetrabutylammonium fluoride (TBAF). Acylation of TBA-alginate was performed under homogeneous conditions, such that both M and G residues were acetylated up to a total degree of substitution (DS) ≈1.0. Performing the same reaction under heterogeneous conditions resulted in selective acylation of M residues. Regioselectivity in the acylated alginate products was studied, and degradation under basic reaction conditions was probed.     

Monolayer adsorption of a "bald" mutant of the highly adhesive and hydrophobic bacterium Acinetobacter sp. strain Tol 5 to a hydrocarbon surface

The affinity of microbial cells for hydrophobic interfaces is important because it directly affects the efficiency of various bioprocesses, including green biotechnologies. The toluene-degrading bacterium Acinetobacter sp. strain Tol 5 has filamentous appendages and a hydrophobic cell surface, shows high adhesiveness to solid surfaces, and self-agglutinates. A "bald" mutant of this bacterium, strain T1, lacks the filamentous appendages and has decreased adhesiveness but retains a hydrophobic cell surface. We investigated the interaction between T1 cells and an organic solvent dispersed in an aqueous matrix. During a microbial-adhesion-to-hydrocarbon (MATH) test, which is frequently used to measure cell surface hydrophobicity, T1 cells adhered to hexadecane droplet surfaces in a monolayer, whereas wild-type cells aggregated on the droplet surfaces. The adsorbed T1 cells on the hexadecane surfaces hindered the coalescence of the droplets formed by vortexing, stabilizing the emulsion phase. Following the replacement of the aqueous phase with fresh pure water after the MATH test, a proportion of the T1 cells that had adsorbed to the hydrocarbon surface detached during further vortexing, suggesting a reversible adsorption of T1 cells. The final ratio of the adhering cells to the total cells in the detachment test coincided with that in the MATH test. The adhesion of T1 cells to the hydrocarbon surface conformed to the Langmuir adsorption isotherm, which describes reversible monolayer adsorption. Reversible monolayer adsorption should be useful for green technologies employing two-liquid-phase partitioning systems and for bioremediation because it allows effective reaction and transport of hydrophobic substrates at oil-water interfaces.     

Silica nanotubes-doped alginate gel for yeast alcohol dehydrogenase immobilization

A novel alginate–silica nanotubes (ALG–SiNTs) composite was prepared through the incorporation of silica nanotubes (SiNTs) into the alginate (ALG) gel followed by Ca²⁺ cross-linking for encapsulating yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) from Saccharomyces cerevisiae. Pre-adsorption of YADH onto the surface of SiNTs before encapsulating in alginate gel was adopted to circumvent the enzyme leakage. AFM and SEM characterization confirmed that YADH molecules were substantially adsorbed on the SiNTs. SEM and EDX studies showed that the SiNTs homogenously distributed in alginate matrix. The enzyme leakage from ALG–SiNTs–YADH composite was remarkably reduced about 50% compared to that of ALG–YADH composite. Meanwhile, the optimum reaction condition, catalytic activity and kinetic parameters of immobilized YADH in ALG–SiNTs composite were studied. The results showed that stronger affinity between substrates and enzyme, higher activity retention, improved storage and operational stability were achieved when YADH was immobilized in ALG–SiNTs composite instead of ALG–YADH composite.     

Isolation and Properties of Alginate Lyase from the Mid-Gut Gland of Wreath Shell Turbo cornutus

Alginate lyase [EC 4.2.2.3] has been purified from mid-gut gland of wreath shell. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. A molecular weight of 32,000 was estimated by SDS-polyacrylamide gel electrophoresis. Absorption spectra of the reaction products indicated that the enzyme had lyase activity. The enzyme was most active at a pH range of about 8.8 to 9.2 and most stable at pH 5 to 6. Phosphate showed strong stabilizing and enhancing effects on the enzyme activity. Divalent cations behaved differently toward alginic acid and propylene glycol alginate, suggesting requirements for free carboxyl groups and a single glycosidic chain in the enzyme action.     

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler¹ in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m²/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO₂ conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO₂ drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm³), high specific surface area (350 - 500 m²/g) and pore volume (3 - 7 cm³/g for pore sizes less than 150 nm).     

Carbohydrates of the antarctic brown seaweed Ascoseira mirabilis

Mannitol, sucrose and four monosaccharides were obtained from an ethanolic extract of Ascoseira mirabilis. Sequential extraction with aqueous calcium chloride, dilute acid and dilute alkali gave mixtures of laminaran, ‘fucan’ and alginic acid. Laminarans fractionated from the extracts contained different proportions of uniformly (1 → 3) and (1 → 6) linked chains of β-D-glucose residues. The ‘fucan’ contained varying proportions of fucose, galactose and glucuronic acid, small amounts of xylose, mannose, glucose, half ester sulphate and protein. Extraction of the weed under mild alkaline conditions gave a yield of 13.4% of low molecular weight calcium alginate with a mannuronate to guluronate ratio of 30:70 and only a small proportion of sequences of alternating residues. Selective extraction and fractionation gave alginate fractions rich (> 80%) in mannuronate or guluronate.     

Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A2 Action at the Single Molecule Level

We monitored the action of phospholipase A₂ (PLA₂) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA₂ molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the buildup of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA₂. The mobility of individual enzymes on the monolayers was characterized by single particle tracking. Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3.2 μm²/s on the L-DPPC and 4.9 μm²/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to ≈0.2 μm²/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between ≈30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e., enzymes were simply reflected from the gel domains back into solution.     

Contribution of Membrane Elastic Energy to Rhodopsin Function

We considered the issue of whether shifts in the metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium from lipid composition are fully explicable by differences in bilayer curvature elastic stress. A series of six lipids with known spontaneous radii of monolayer curvature and bending elastic moduli were added at increasing concentrations to the matrix lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the MI-MII equilibrium measured by flash photolysis followed by recording UV-vis spectra. The average area-per-lipid molecule and the membrane hydrophobic thickness were derived from measurements of the ²H NMR order parameter profile of the palmitic acid chain in POPC. For the series of ethanolamines with different levels of headgroup methylation, shifts in the MI-MII equilibrium correlated with changes in membrane elastic properties as expressed by the product of spontaneous radius of monolayer curvature, bending elastic modulus, and lateral area per molecule. However, for the entire series of lipids, elastic energy explained the shifts only partially. Additional contributions correlated with the capability of the ethanolamine headgroups to engage in hydrogen bonding with the protein, independent of the state of ethanolamine methylation, with introduction of polyunsaturated sn-2 hydrocarbon chains, and with replacement of the palmitic acid sn-1 chains by oleic acid. The experiments point to the importance of interactions of rhodopsin with particular lipid species in the first layer of lipids surrounding the protein as well as to membrane elastic stress in the lipid-protein domain.     

Enzymes involved in the biosynthesis of alginate byPseudomonas aeruginosa

Summary Analysis of the enzymes involved in the biosynthesis of alginic acid by mucoidPseudomonas aeruginosa PAO strain's determined the presence of enzymes required to synthesise GDP-mannuronic acid. Addition of polymannuronic acid to an ammonium sulphate precipitate of a cell free alginate suspension indicated the presence of an enzyme which catalysed the epimerisation of mannuronic acid to guluronic acidafter the polymer had been synthesised. The epimerase was shown to be calcium dependant.Various non-mucoid mutants were also studied. The non-mucoid parental strain PAO 381 also contained the enzymes required for alginate synthesis but they were not expressed. Synthesis of alginic acid led to an increase in the level of these enzymes. In the non-mucoid mutants derived from mucoid parents GDP-mannose dehydrogenase was absent in all strains studied. In some of these strains GDP-mannose pyrophosphorylase was also absent, while in other strains, phosphomannase isomerase was absent or greatly reduced.