Recovery of astaxanthin using colloidal gas aphrons (CGA): a mechanistic study

The aim of this study is to investigate the mechanism responsible for the recovery of astaxanthin using Colloidal Gas Aphrons (CGA), which are surfactant stabilised microbubbles. The latter were produced using different surfactant solutions (Cetyl Trimethyl Ammonium Bromide (CTAB)-cationic, Sodium Dodecyl Sulfate (SDS)-anionic, TWEEN 60-non-ionic and mixtures of TWEEN 60-SPAN 80- non-ionic with varying hydrophobicity) at stirring speed 8000 rpm and stirring time 5 min. Experiments were carried out at varying pH and volumetric ratios of astaxanthin to CGA, and with two different astaxanthin standard suspensions: (i) astaxanthin dispersed in aqueous solutions and (ii) astaxanthin dispersed in ethanolic/aqueous solutions with different compositions of ethanol (20/80 (v/v) and 40/60 (v/v)). When astaxanthin is dispersed in aqueous solutions the separation seems to occur mainly by electrostatic interactions. Therefore the recoveries are higher in the case of the cationic surfactant when astaxanthin particles are strongly negatively charged, as shown by the zeta potential measurements. When ethanol is present, highest recoveries are achieved with CGA produced from the non-ionic surfactant, which indicates that, under these conditions, separation is driven mainly by hydrophobic interactions. In experiments with ethanolic/aqueous suspensions, when the hydrophobicity of the surfactant was increased by increasing volumes of SPAN 80, the CGA produced were less stable; thus higher recoveries of astaxanthin under conditions that favour hydrophobic interactions were not observed. (C) 2008 Elsevier B.V All rights reserved.

Description of Enterococcus canis sp nov from dogs and reclassification of Enterococcus porcinus Teixeira et al. 2001 as a junior synonym of Enterococcus villorum Vancanneyt et al. 2001

Strains from anal swabs and chronic otitis externa in dogs were shown to be phylogenetically related to the Enterococcus faecium species group. They shared a number of phenotypic characteristics with these species, but they could be easily differentiated by biochemical reactions. In addition, the canine strains were unusual in their nearly complete failure to grow on sodium azide-containing enterococci-selective media and in their Voges-Proskauer reactions (usually negative). By using 16S rRNA sequencing and DNA-DNA hybridization of representative strains, as well as tDNA interspacer gene PCR and SDS-PAGE of whole-cell proteins, the group of canine strains was shown to constitute a novel enterococcal species. The name Enterococcus canis sp. nov. is proposed for this species, with LMG 12316(T) (= CCUG 46666(T)) as the type strain. Concurrently, the taxonomic situation and nomenclatural position of Enterococcus porcinus were investigated. As no phenotypic or genotypic differences were found between this species and Enterococcus villorum, the name E. porcinus is considered to be a junior synonym of E. villorum.

Streptococcus devriesei sp nov., from equine teeth

Phenotypic and phylogenetic studies were performed on four unidentified Gram-positive staining, catalase-negative, cc-hemolytic Streptococcus-like organisms recovered from the teeth of horses. SDS PAGE analysis of whole-cell proteins and comparative 16S rRNA gene sequencing demonstrated the four strains were highly related to each other but that they did not correspond to any recognised species of the genus Streptococcus. Phylogenetic analysis based on 16S rRNA gene sequences showed the unidentified organisms form a hitherto unknown sub-line within the Streptococcus genus, displaying a close affinity with Streptococcus mutans, Streptococcus ferus and related organisms. Sequence divergence values of > 5% with thew and other reference streptococcal species however demonstrated the organisms from equine sources represent a novel species. Based on the phenotypic distinctiveness of the new bacterium and molecular chemical and molecular genetic evidence, it is proposed that the unknown species be classified as Streptococcus devriesei sp. nov. The type strain of Streptococcus devriesei is CCUG 47155(T) (= CIP 107809T).

Some functional properties of fractionated soy protein isolates obtained by microfiltration

Five soy proteins isolate (SPI) fractions were produced using two microfiltration membranes with different pore sizes. Fractionation was carried out on SPI produced by isoelectric precipitation of a crude protein extract. The five fractions were two retentates and two permeates from the two membranes, the fifth fraction was obtained as the retentate on the smaller-po re- sized membrane fed with the permeate from the larger-pore-sized membrane. Solubility, foaming and emulsifying properties of the collected fractionates were investigated. It was observed that in the pH range 3-8 the retentates featured superior solubility compared with permeates. There was no significant difference (p > 0.0 1) in solubility between the retentates and SPI at pH >= 6. Foaming characteristics of the fractions followed the same trend as solubility with regard to foam expansion. There was, however, no particular trend observed with regards to foam stability. Emulsions stabilised by the retentates exhibited higher values (p<0.01) of emulsion stability index (ESI) and emulsifying activity index (EAI) than those stabilised with permeates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles indicated that the fractions exhibiting high functionality in terms of solubility, foaming and emulsifying properties were also richer in 7S globulin soy protein subunits. Isoelectric focussing (IEF) profiles showed that retentates were richer in species with isoelectric points (pl) between 5.2 and 5.6 while permeates featured more prominently at pis between 4.5 and 4.8. (C) 2006 Elsevier Ltd. All rights reserved.

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillits phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5. a K-m of 14 mM with alpha-D-Manp-(1 -> 6)-D-Manp as substrate. It was strongly inhibited by Mn2+ and did not need Ca2+ or any other metal cofactor of those tested. The enzyme cleaves specifically (1 -> 6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocus sing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1 -> 3)-D-Manp 10 times faster than alpha-D-Manp-(1 -> 6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1 -> 2)-D-Manp. The activity towards (1 -> 3)-linked mannobiose is strongly activated by 1 mM Ca2+ and inhibited by 10 mM EDTA, while (1 -> 6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities. (c) 2005 Elsevier Ltd. All rights reserved.

Isolation, fractionation and characterisation of proteins from Mucuna bean

The chemical composition and fractional distribution of protein isolates prepared from species of Mucuna bean were studied. Using six different extraction media, the yield of protein based on the Kjeldahl procedure varied from 8% to 34%, and the protein content varied from 75% to 95%. When the yields were high, the colour of the isolates generally tended to be dark and unsatisfactory. Hence, the use of chemical treatments and high pressure processing were explored. The solubility maxima for the protein isolates in water were found to occur at pH values of 2.0 and 11.0, while the pH corresponding to minimum solubility (i.e. isoelectric region) occurred at pH values of 4.0 and 5.0. The total essential amino acid in the isolates ranged from 495 to 557 mg g(-1) protein, which compares favourably with the recommended level for pre-school and school children. Methionine and cysteine were the limiting amino acids. A key nutritional attribute of the protein isolates was its high lysine content. The isolate can therefore complement cereal-based foods which are deficient in lysine. The proteins mainly consisted of albumins, glutelins and globulins. Prolamins were only present in trace concentration (< 0.3%). Gel filtration chromatograms of the isolates indicated the presence of major protein fractions with molecular weights of 40 and 15 kDa, while gel electrophoresis (SDS-PAGE) indicated a major broad zone with molecular weights of 36 +/- 7 and 17.3 +/- 13 kDa. (c) 2006 Elsevier Ltd. All rights reserved.

Stabilizing swarm intelligence search via positive feedback resource allocation

A novel Swarm Intelligence method for best-fit search, Stochastic Diffusion Search, is presented capable of rapid location of the optimal solution in the search space. Population based search mechanisms employed by Swarm Intelligence methods can suffer lack of convergence resulting in ill defined stopping criteria and loss of the best solution. Conversely, as a result of its resource allocation mechanism, the solutions SDS discovers enjoy excellent stability.

Methods for comparing and constraining models of dendritic spines

Physiological evidence using Infrared Video Microscopy during the uncaging of glutamate has proven the existence of excitable calcium ion channels in spine heads, highlighting the need for reliable models of spines. In this study we compare the three main methods of simulating excitable spines: Baer & Rinzel's Continuum (B&R) model, Coombes' Spike-Diffuse-Spike (SDS) model and paired cable and ion channel equations (Cable model). Tests are done to determine how well the models approximate each other in terms of speed and heights of travelling waves. Significant quantitative differences are found between the models: travelling waves in the SDS model in particular are found to travel at much lower speeds and sometimes much higher voltages than in the Cable or B&R models. Meanwhile qualitative differences are found between the B&R and SDS models over realistic parameter ranges. The cause of these differences is investigated and potential solutions proposed.

Minimum stable convergence criteria for stochastic diffusion search

An analysis of Stochastic Diffusion Search (SDS), a novel and efficient optimisation and search algorithm, is presented, resulting in a derivation of the minimum acceptable match resulting in a stable convergence within a noisy search space. The applicability of SDS can therefore be assessed for a given problem.

Effect of headgroup size, charge, and solvent structure on polymer−micelle interactions, studied by molecular dynamics simulations

We performed atomistic molecular dynamics simulations of anionic and cationic micelles in the presence of poly(ethylene oxide) (PEO) to understand why nonionic water-soluble polymers such as PEO interact strongly with anionic micelles but only weakly with cationic micelles. Our micelles include sodium n-dodecyl sulfate (SDS), n-dodecyl trimethylammonium chloride (DTAC), n-dodecyl ammonium chloride (DAC), and micelles in which we artificially reverse the sign of partial charges in SDS and DTAC. We observe that the polymer interacts hydrophobically with anionic SDS but only weakly with cationic DTAC and DAC, in agreement with experiment. However, the polymer also interacts with the artificial anionic DTAC but fails to interact hydrophobically with the artificial cationic SDS, illustrating that large headgroup size does not explain the weak polymer interaction with cationic micelles. In addition, we observe through simulation that this preference for interaction with anionic micelles still exists in a dipolar "dumbbell" solvent, indicating that water structure and hydrogen bonding alone cannot explain this preferential interaction. Our simulations suggest that direct electrostatic interactions between the micelle and polymer explain the preference for interaction with anionic micelles, even though the polymer overall carries no net charge. This is possible given the asymmetric distribution of negative charges on smaller atoms and positive charges oil larger units in the polymer chain.

Molecular dynamics simulation of interactions between a sodium dodecyl sulfate micelle and a poly(ethylene oxide) polymer

We have performed atomistic molecular dynamics simulations of an anionic sodium dodecyl sulfate (SDS) micelle and a nonionic poly(ethylene oxide) (PEO) polymer in aqueous solution. The micelle consisted of 60 surfactant molecules, and the polymer chain lengths varied from 20 to 40 monomers. The force field parameters for PEO were adjusted by using 1,2-dimethoxymethane (DME) as a model compound and matching its hydration enthalpy and conformational behavior to experiment. Excellent agreement with previous experimental and simulation work was obtained through these modifications. The simulated scaling behavior of the PEO radius of gyration was also in close agreement with experimental results. The SDS-PEO simulations show that the polymer resides on the micelle surface and at the hydrocarbon-water interface, leading to a selective reduction in the hydrophobic contribution to the solvent-accessible surface area of the micelle. The association is mainly driven by hydrophobic interactions between the polymer and surfactant tails, while the interaction between the polymer and sulfate headgroups on the micelle surface is weak. The 40-monomer chain is mostly wrapped around the micelle, and nearly 90% of the monomers are adsorbed at low PEO concentration. Simulations were also performed with multiple 20-monomer chains, and gradual addition of polymer indicates that about 120 monomers are required to saturate the micelle surface. The stoichiometry of the resulting complex is in close agreement with experimental results, and the commonly accepted "beaded necklace" structure of the SDS-PEO complex is recovered by our simulations.

c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis

The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-x(L). Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-x(L) the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-x(L) as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Deltapsim) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Deltapsim and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-x(L) may represent a potential of the modulation of mitochondrial function during apoptosis.

Resource allocation analysis of the stochastic diffusion search

The Stochastic Diffusion Search (SDS) was developed as a solution to the best-fit search problem. Thus, as a special case it is capable of solving the transform invariant pattern recognition problem. SDS is efficient and, although inherently probabilistic, produces very reliable solutions in widely ranging search conditions. However, to date a systematic formal investigation of its properties has not been carried out. This thesis addresses this problem. The thesis reports results pertaining to the global convergence of SDS as well as characterising its time complexity. However, the main emphasis of the work, reports on the resource allocation aspect of the Stochastic Diffusion Search operations. The thesis introduces a novel model of the algorithm, generalising an Ehrenfest Urn Model from statistical physics. This approach makes it possible to obtain a thorough characterisation of the response of the algorithm in terms of the parameters describing the search conditions in case of a unique best-fit pattern in the search space. This model is further generalised in order to account for different search conditions: two solutions in the search space and search for a unique solution in a noisy search space. Also an approximate solution in the case of two alternative solutions is proposed and compared with predictions of the extended Ehrenfest Urn model. The analysis performed enabled a quantitative characterisation of the Stochastic Diffusion Search in terms of exploration and exploitation of the search space. It appeared that SDS is biased towards the latter mode of operation. This novel perspective on the Stochastic Diffusion Search lead to an investigation of extensions of the standard SDS, which would strike a different balance between these two modes of search space processing. Thus, two novel algorithms were derived from the standard Stochastic Diffusion Search, ‘context-free’ and ‘context-sensitive’ SDS, and their properties were analysed with respect to resource allocation. It appeared that they shared some of the desired features of their predecessor but also possessed some properties not present in the classic SDS. The theory developed in the thesis was illustrated throughout with carefully chosen simulations of a best-fit search for a string pattern, a simple but representative domain, enabling careful control of search conditions.

Convergence analysis of stochastic diffusion search

In this paper we present a connectionist searching technique - the Stochastic Diffusion Search (SDS), capable of rapidly locating a specified pattern in a noisy search space. In operation SDS finds the position of the pre-specified pattern or if it does not exist - its best instantiation in the search space. This is achieved via parallel exploration of the whole search space by an ensemble of agents searching in a competitive cooperative manner. We prove mathematically the convergence of stochastic diffusion search. SDS converges to a statistical equilibrium when it locates the best instantiation of the object in the search space. Experiments presented in this paper indicate the high robustness of SDS and show good scalability with problem size. The convergence characteristic of SDS makes it a fully adaptive algorithm and suggests applications in dynamically changing environments.

Quantitation of apolipoprotein B-48 in triacylglycerol-rich lipoproteins by a specific enzyme-linked immunosorbent assay

This paper describes the use of an antiserum, specific for apolipoprotein (apo) B-48, in a competitive, enzyme-linked immunosorbent assay (ELISA) for apo B-48 in triacylglycerol-rich lipoprotein (TRL) fractions prepared from fasting and post-prandial plasma samples. Previously we showed the antiserum to act as an effective immunoblotting agent following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its use in this ELISA indicates that the antiserum recognises the C-terminal region of the protein on the surface of lipoprotein particles. The ELISA had a sensitivity of less than 37 ng/ml and intra- and inter-assay coefficients of variation of 3.8% and 8.6%, respectively. There was no cross-reaction in the ELISA against serum albumin, ovalbumin, thyroglobulin, or apo B-100 (purified by immunoaffinity chromatography), and high lipid concentrations (as Intralipid) did not interfere. A low density lipoprotein fraction reacted in the ELISA but SDS-PAGE-Western blot analysis confirmed the presence, in the fraction, of a small amount of apo B-48, indicating the existence of low density dietary-derived lipoprotein particles. ELISA and SDS-PAGE-Western blot analysis were used to measure apo B-48 in 12 series of postprandial samples collected from 4 diabetic and 8 normal subjects, following test meals of varying fat content. The mean correlation between the two methods was r = 0.74. The mean fasting concentration of apo B-48 in the TRL fractions from 15 healthy men was 0.46 μg/ml of plasma.