Speciation analysis,bioavailability and risk assessment of trace metals in herbal decoctions using a combined technique of in Vitro digestion and biomembrane filtration as sample pretreatment method

Sample preparation is the first crucial step in the speciation analysis, bioavailability and risk assessment of trace metals in plant samples such as herb and vegetables. Two bionic technologies titled ‘in vitro digestion’ and ‘extraction with biomembrane’ were developed for pre‐treatment of herbal decoction. The decoctions of Aconiteum carmichaeli and Paeonia lactiflora were digested at body temperature, at the acidity of the stomach or intestine and with inorganic and organic materials (digestive enzymes were included for whole‐bionic and excluded for semi‐bionic) found in the stomach or intestine. Being similar to the biomembrane between the gastrointestinal tract and blood vessels, monolayer liposome was used as a biomembrane model. Affinity‐monolayer liposome metals and water‐soluble metals were used for speciation analysis and bioavailability assessment of copper and zinc in herbal decoction. In the decoction of Aconiteum carmichaeli and Paeonia lactiflora, Zn was mainly absorbed in the intestine and Cu was mainly absorbed by both stomach and intestine. The safe dosage for males and females is below 257.1 g/day Aconiteum carmichaeli and 529.4 g/day Paeonia lactiflora. Copyright © 2010 John Wiley & Sons, Ltd.     

Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D₂O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D₂O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.     

Microstructure of Purified Rubber Particles

Purified rubber particles from Hevea brasiliensis (Brazilian rubber tree), Parthenium argentatum (guayule), Ficus elastica (Indian rubber tree), and Euphorbia lactiflua were examined and compared using conventional scanning electron microscopy (SEM), field-emission SEM, cryo-SEM, and transmission electron microscopy (TEM). Rubber particles of all four species were spherical; they varied in size and had a uniform homogeneous material, the rubber core, surrounded by a contiguous monolayer (half-unit) membrane. Frozen-hydrated and/or untreated particles from H. brasiliensis and P. argentatum deformed and fused readily, whereas those from F. elastica and E. lactiflua retained their spherical shapes. These results indicate that the surface components of the H. brasiliensis and P. argentatum particles are more fluid than those of F. elastica or E. lactiflua. When fixed in aldehyde, F. elastica particles retained their spherical exterior shapes but had hollow centers, whereas H. brasiliensis and P. argentatum particles completely collapsed. In aldehyde-osmium tetroxide-fixed material, the rubber core of F. elastica was poorly preserved in some particles in which only a small amount of the rubber core remained adhering to the monolayer membrane, leaving a hollow center. Euphorbia lactiflua particles were well preserved in terms of retaining the rubber core; however, the membrane was not as easily discernible as it was in the other three species. Both H. brasiliensis and P. argentatum were well preserved following fixation; their cores remained filled with rubber, and their monolayer membranes were defined. The addition of potassium permanganate to the fixation-staining regime resulted in higher-contrast micrographs and more well defined monolayer membranes.     

Bacterial adhesion to different termite flagellates: Ultrastructural and functional evidence for distinct molecular attachment modes

Summary The attachment modes of rodlike ectobiotic bacteria to the surface of two different termite flagellates were studied.Devescovina glabra was covered by laterally attached bacteria. Treatment with chemicals that disturb hydrophobic interactions and solubilize proteins removed the ectobionts. Freeze-fracture and freeze-etching electron microscopy revealed rows of intramembrane particles that occurred exclusively along the attachment sites. The adhering Gram-negative bacteria possessed an S-layer (surface layer) composed of globular protein particles. The S-layer could be removed by protein-solubilizing chemicals, e.g., urea, as shown by ultrathin-section electron microscopy. Therefore, it seems plausible that the attachment was mediated by hydrophobic interactions between the flagellate's plasma membrane and the S-layer of the bacteria. The bacteria of the second flagellate,Joenia annectens, adhered by their tips. The attachment was extremely strong. Chemicals disturbing ionic or hydrophobic bindings or solubilizing proteins did not detach the ectobionts. Globular intramembrane protein particles were preferentially found in a ringlike array at the external fracture face of the flagellate's contact sites.Abbreviations DIC differential interference contrast - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - TEM transmission electron microscope - Tween 20 polyoxyethylenesorbitan     

Stabilization of the Bio-membrane by Small Molecules: Interaction of Trehalose with the Phospholipid Bilayer

Abstract

Anhydrobiotic organisms undergo periods of acute dehydration during their life cycle. It is of interest to understand how the biomembrane remains intact through such stress. A di-saccharide, trehalose, which is metabolised during anhydrobiosis is found to prevent disruption of model membrane systems (1). Molecular modelling techniques are used to investigate the possible mode of interaction of trehalose with a model monolayer. The objective is to maximise hydrogen bonding between the two systems. A phospholipid matrix consisting of l,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) is chosen to represent the monolayer. The crystal structure of DMPC (2) reveals that there are two distinct conformers designated as A and B. An expansion of the monolayer, coplanar with its surface, results in the trehalose molecule being accomodated in a pocket formed by four B conformers. One glucose ring of the sugar rests on the hydrophobic patch provided by the choline methyls of an A conformer. Five hydrogen bonds are formed involving the phosphate oxygens of three of the surrounding B conformers. The model will be discussed with reference to relevant experimental data on the interaction.     

The Formation of g=2.49-Species of Cytochrome P450 in the Rat Liver by PCB126 Oral Administration: Identification of Heme Axial Ligands by EPR Spectroscopy

Rat livers and microsomes were subjected to electron paramagnetic resonance (EPR) measurements at 77 K. The EPR spectra of the livers from the control group, carbon tetrachloride-, 3-methylcholanthrene-, and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126)-treated rats exhibited an EPR spectrum at g=2.40, 2.24, and 1.93, which is characteristic of P450 in a resting state. The liver of the PCB126-treated rats showed an additional distinct EPR spectrum at g=2.49, 2.26, and 1.87 (g=2.49-species). The heme environmental structure of g=2.49-species was identified by crystal field analysis using three EPR g-values of the microsome treated with various chemicals. These results indicated that g=2.49-species is a hemeprotein with cysteine thiolate at the 5th coordination site, and a nitrogenous ligand at the 6th site.     

Insertion of lipidated Ras proteins into lipid monolayers studied by infrared reflection absorption spectroscopy (IRRAS)

Ras proteins have to be associated with the inner leaflet of the plasma membrane to perform their signaling functions. This membrane targeting and binding is controlled by post-translational covalent attachment of farnesyl and palmitoyl chains to cysteines in the membrane anchor region of the N- and H-Ras isoforms. Two N-Ras lipoproteins were investigated, namely a farnesylated and hexadecylated protein, presenting the natural hydrophobic modifications and a doubly hexadecylated construct, respectively. The proteins are surface active and form a Gibbs monolayer at the air-D2O interface. The contours of the amide-I bands were analyzed using infrared reflection absorption spectroscopy (IRRAS). Langmuir monolayers of a mixture of POPC, brain sphingomyelin, and cholesterol were used as half of a model biomembrane to study the insertion of these N-Ras proteins. They insert with their hydrophobic anchors into lipid monolayers but at higher surface pressures (30 mN/m); the farnesylated and hexadecylated protein desorbs completely from the monolayer, whereas the doubly hexadecylated protein remains incorporated. During the insertion process, changes in the orientation of the protein secondary structure were detected by comparison with simulated IRRA spectra, based on the information on the relative orientation of the secondary structure elements from the protein crystal structure data.     

Spreading of Bacillus thuringiensis var. israelensis over the water surface by a monolayer carrier

The self-spreading properties of monolayers, acting as carriers for Bacillus thuringlensis var. israelensis, were examined in the laboratory. Drops of mixtures of 0.5 to 5% B. thuringlensis in a liquid monolayer were placed at one end of a narrow static water course, 2 m long. In less than 2 min, a single layer of molecules spread over the water surface and deposited enough B. thuringiensis over the whole 2 m to kill test mosquito larvae in 24 h. The monolayer alone did not kill larvae in 24 h because no vegetation was present to deoxygenate the water overnight. Accelerated biodegradation was simulated carrying surface water into an overflow tank for 6 h, the surface monolayer being replenished until the mixture was exhausted. B. thuringiensis was deposited in decreasing quantity along the course with much reaching the overflow tank. The monolayer can carry B. thuringiensis more than 15 m in the field from a point application, dispensing with the need for spray equipment, particularly relevant in developing countries.The author is with the Institute of Horticultural Research, Worthing Road, Littlehampton, West Sussex BN17 6LP, UK.     

Magnetoliposomes

The adsorption of different types of phosphatidylglycerols onto magnetizable solid particles is studied. The super-paramagnetic magnetite spheres used have an average diameter of only 14 nm and are stabilized by lauric acid to keep them in solution. During incubation and dialysis of this water-based magnetic fluid in the presence of preformed sonicated phospholipid vescles, magnetoliposomes are formed which are captured from solution with high efficiency by high-gradient magnetophoresis. Support for the bilayer character of the phospholipid coat is derived from both theoretical calculations and experimental data. Phospholipids which form the inner monolayer are adsorbed very quickly with their charged headgroup orientated towards the iron oxide surface. The high-affinity character of the binding is reflected in the adsorption isotherms and is further illustrated by their non-extractability with high concentrations of Tween 20. The outer layer assembles through interaction with the exposed hydrocarbon chains. As compared to the inner layer, the phospholipids adsorb at a much slower rate and are displaced by Tween 20 concentrations which usually disrupt conventional membranes. The adsorption isotherms for this layer obey the Langmuir expression. The affinity constants, derived from them, progressively increase as the hydrophobic nature of the phosphatidylglycerols is more pronounced.Abbreviations DXPG di-fatty acyl form of phosphatdylglycerol where X=L, Lauroyl - M myristoyl - C _15:0 pentadecanoyl - O oleoyl. TES, 2-((tris(hydroxymethyl)methyl)amino)ethanesulfonic acid     

The biosynthesis of rubber: Incorporation of isopentenyl pyrophosphate into purified rubber particles by a soluble latex serum enzyme

1. The rubber particles in Hevea brasiliensis latex have been partially purified by `washing' with buffer solution, and separated into active fractions of different particle size. 2. The enzyme responsible for incorporating isopentenyl pyrophosphate into rubber is distributed between the surface of the rubber particles and the aqueous serum phase of the latex. The enzyme at the surface can be removed or inactivated if the rubber particles are washed sufficiently with buffer solution. Enzyme in the serum phase can be concentrated by fractional precipitation with ammonium sulphate. 3. To incorporate isopentenyl pyrophosphate into rubber in vitro, active rubber particles are required as well as enzyme and soluble cofactors. The activity of the rubber particles per unit surface area increases with diminishing particle size.     

Mycobacterium abscessus cell wall and plasma membrane characterization by EPR spectroscopy and effects of amphotericin B,miltefosine and nerolidol

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to characterize the components of the Mycobacterium abscessus massiliense cell envelope and their interactions with amphotericin B (AmB), miltefosine (MIL), and nerolidol (NER). Spin labels analogous to stearic acid and phosphatidylcholine (PC) were distributed on an envelope layer with fluidity comparable to other biological membranes, probably the mycobacterial cell wall, because after treatment with AmB a highly rigid spectral component was evident in the EPR spectra. Methyl stearate analogue spin labels found a much more fluid membrane and did not detect the presence of AmB, except for at very high drug concentrations. Unlike other spin-labeled PCs, the TEMPO-PC spin probe, with the nitroxide moiety attached to the choline of the PC headgroup, also did not detect the presence of AmB. On the other hand, the steroid spin labels were not distributed across the membranes of M. abscessus and, instead, were concentrated in some other location of the cell envelope. Both MIL and NER compounds at 10 μM caused increased fluidity in the cell wall and plasma membrane. Furthermore, NER was shown to have a remarkable ability to extract lipids from the mycobacterial cell wall. The EPR results suggest that the resistance of mycobacteria to the action of AmB must be related to the fact that this drug does not reach the bacterial plasma membrane.     

Bacterial scavenging: Utilization of fatty acids localized at a solid-liquid interface

A model oligotrophic aquatic system involving localization of fatty acids on a solid surface was used to quantitate scavenging by three bacteria; Leptospira biflexa patoc 1 which adheres reversibly, pigmented Serratia marcescens EF190 which adheres irreversibly, and a non-pigmented hydrophilic mutant of EF190. The Leptospira and pigmented Serratia displayed two distinct scavenging strategies which are related to their different methods of adhesion. The Leptospira efficiently scavenged [1-¹⁴C] stearic acid from the surface in 24 h, whereas the pigmented hydrophobic Serratia initially showed a faster rate of removal but the overall rate was considerably slower than that of the Leptospira. The hydrophilic, non-pigmented Serratia required 50h incubation to remove significant amounts of the labelled fatty acid. The greater scavenging ability of the hydrophobic pigmented Serratia strain compared to the hydrophilic non-pigmented mutant could not be attributed to differences in viability of fatty acid metabolism. The hydrophobicity of the pigmented Serratia allows for firmer adhesion and greater interaction with the surface localized nutrients.     

Identification of a Taraxacum brevicorniculatum rubber elongation factor protein that is localized on rubber particles and promotes rubber biosynthesis

Two protein families required for rubber biosynthesis in Taraxacum brevicorniculatum have recently been characterized, namely the cis‐prenyltransferases (TbCPTs) and the small rubber particle proteins (TbSRPPs). The latter were shown to be the most abundant proteins on rubber particles, where rubber biosynthesis takes place. Here we identified a protein designated T. brevicorniculatum rubber elongation factor (TbREF) by using mass spectrometry to analyze rubber particle proteins. TbREF is homologous to the TbSRPPs but has a molecular mass that is atypical for the family. The promoter was shown to be active in laticifers, and the protein itself was localized on the rubber particle surface. In TbREF‐silenced plants generated by RNA interference, the rubber content was significantly reduced, correlating with lower TbCPT protein levels and less TbCPT activity in the latex. However, the molecular mass of the rubber was not affected by TbREF silencing. The colloidal stability of rubber particles isolated from TbREF‐silenced plants was also unchanged. This was not surprising because TbREF depletion did not affect the abundance of TbSRPPs, which are required for rubber particle stability. Our findings suggest that TbREF is an important component of the rubber biosynthesis machinery in T. brevicorniculatum, and may play a role in rubber particle biogenesis and influence rubber production.     

Selective isolation of bacteria from soil with hydrophobic materials

Bacterial strains having a hydrophobic cell surface have often been considered as degraders of hydrophobic organic pollutants in soil. In this study, bacterial strains were isolated using hydrophobic materials from 12 soil samples, and their cell surface hydrophobicity was determined by evaluating their adherence to n-hexane. Bacterial strains isolated using polytetrafluoroethylene (PTFE) membrane were more hydrophobic on an average than those isolated with styrene–divinylbenzene (DVB) particles or octadecylsilyl silica gel (ODS) particles. Strains closely related to Burkholderia cepacia could be selectively isolated using the PTFE membrane; those closely related to Ralstonia pickettii, using ODS and DVB particles; and those closely related to B. fungorum, using DVB. These results indicate that bacterial strains having a hydrophobic cell surface or within certain phyla can be selectively isolated from soils using hydrophobic materials, and that this isolation method would be useful for collecting candidates for bioremediation of hydrophobic pollutants.     

Purity of the sacred lotus, or escape from contamination in biological surfaces

The microrelief of plant surfaces, mainly caused by epicuticular wax crystalloids, serves different purposes and often causes effective water repellency. Furthermore, the adhesion of contaminating particles is reduced. Based on experimental data carried out on microscopically smooth (Fagus sylvatica L., Gnetum gnemon L., Heliconia densiflora Verlot, Magnolia grandiflora L.) and rough water-repellent plants (Brassica oleracea L., Colocasia esculenta (L.) Schott., Mutisia decurrens Cav., Nelumbo nucifera Gaertn.), it is shown here for the first time that the interdependence between surface roughness, reduced particle adhesion and water repellency is the keystone in the self-cleaning mechanism of many biological surfaces. The plants were artificially contaminated with various particles and subsequently subjected to artificial rinsing by sprinkler or fog generator. In the case of water-repellent leaves, the particles were removed completely by water droplets that rolled off the surfaces independent of their chemical nature or size. The leaves of N. nucifera afford an impressive demonstration of this effect, which is, therefore, called the “Lotus-Effect” and which may be of great biological and technological importance. Received: 19 August 1996 / Accepted: 12 November 1996     

Ultrastructural changes during fertilization envelope assembly in Lytechinus pictus eggs revealed by quick-freeze,deep-etch electron microscopy

Morphological features of fertilization envelope assembly in egges from the sea urchin Lytechinus pictus were examind in platinum replicas of samples quick-frozen, deep-etched, and rotary-shadowed at various times after insemination. Unfertilized eggs are surrounded by the vitelline layer, a glycocalyx, which faith-fully follows the contours of the microvillus-studded egg surface. The vitelline layer is secured to the plasma membrane below via a series of short projections called vitelline posts. The vitelline matrix itself is an elaborate meshwork of uniformly sized filaments, which are decorated in places with globular particles. At fertilization, the vitelline layer elevates off the egg surface and by 1 min after insemination appears as a thin, airy network of fibers. In contrast to Strongylocentrotus purpuratus, impressions of the underlying microvilli are not retained in this species. The vitelline template appears to become filled in by the deposition of amorphous secretory material between 1 and 5 min after fertilization. This smooth, amorphous layer is then coated with a thin sheet of paracrystalline material. Paracrystalline coating is incomplete at 5 min, but by 20 min after insemination the coat is complete, consisting of ordered parallel rows of roset-telike particles.     

The Structure of Mycoplasma pneumoniae as Determined by the Freeze-Substitution Technique

The ultrastructure of Mycoplasma pneumoniae FH was examined by a mild fixation method, the freeze-substitution technique, for thin-section transmission electron microscopy and the following new findings were obtained. In the cytoplasm, no nuclear region could be clearly identified. The cytoplasm was filled with many ribosome-like particles, fine fibers and electron-dense particles. The electron-dense particles appeared to be similar to the particles found in the nucleoid region of Pseudomonas aeruginosa and might therefore possibly be a kind of DNA binding protein. The cell surface was completely enveloped with a thin opaque layer. The presence of this surface layer prevented any direct contact of the cell surface with that of the two M. pneumoniae cells.     

Specific binding of peanut agglutinin to GM1-doped solid supported lipid bilayers investigated by shear wave resonator measurements

This study deals with the specific interaction between the lectin peanut agglutinin (PNA) from Arachis hypogaea and the ganglioside G_M1 which was incorporated in a solid supported lipid bilayer immobilized on a gold electrode placed on top of an AT-cut quartz crystal. Bilayer formation was reached by self-assembly processes. The first monolayer consists of octanethiol attached to the gold surface via chemisorption and the second monolayer was immobilized by vesicle fusion on the preformed hydrophobic surface. We managed to keep unspecific binding to a minimum by using a phospholipid matrix consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Lectin binding to ganglioside G_M1 containing membranes was determined by a decrease of the resonant frequency of the quartz crystal. The minimum amount of receptor within the membrane which is necessary to obtain a complete protein monolayer was found to be less than 2 mol%. The adsorption isotherm of PNA to G_M1 was recorded and analyzed to be of Langmuir type, exhibiting a binding constant of PNA to the ganglioside of 8.3 ⋅ 10⁵ M^–1. The good agreement of the calculated Langmuir adsorption isotherm with the obtained experimental data implies that protein multilayers are not formed and that interactions between the adsorbents can be neglected. Furthermore, the association constants of two different saccharides, β-Galp-(1 → 3)-GalNAc exhibiting a strong binding to PNA in solution, and β-D-galactose with a much lower affinity were estimated by determining the equilibrium concentration of PNA attached to the surface. Moreover we were able to remove the attached lectin monolayer by digestion of the protein with pronase causing an increase in the resonant frequency which almost reversed the frequency shift to lower frequencies during adsorption. An even more complex system was built up by the use of digoxigenin-labeled PNA which also binds to the solid supported membrane containing the receptor G_M1. The immobilized lectin was recognized by anti-digoxigenin-F_ab-fragments, which is measurable by a further decrease of the resonant frequency. For all binding processes we found larger frequency shifts for a complete protein monolayer than predicted by Sauerbrey's equation, clearly showing that in addition to mass loading viscoelastic changes occur at the lipid-protein interface. Received: 22 July 1996 / Accepted: 12 September 1996     

Self-assembly of two-patch particles in solution: a Brownian dynamics simulation study

We study the self-assembly behaviour of two-patch particles with D_∞h symmetry by using Brownian dynamics simulations. The self-assembly process of two-patch particles with diverse patch coverage in two selective solvent conditions is investigated. The patchy particles in a solvent that is bad for patches but good for matrix form linear thread-like structures with low patch coverage, whereas they form 3D network structures with relatively high patch coverage on surface. For patchy particles in a solvent which is good for patches but bad for body, monolayer structures are obtained at high patch coverage, and some cluster structures emerge when surface patch coverage is low.     

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz – role of cis‐prenyltransferase‐like 1 protein

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high‐molecular‐weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis‐1,4‐isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis‐prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT‐like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis‐1,4‐isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1‐RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low‐expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS‐based comparison of latex proteomes from TkCPTL1‐RNAi plants and T. koksaghyz wild‐types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.