Effect of lutein on the transport of Ca2+ across phospholipid bilayer and mitochondrial membrane

Lutein (3,3'-dihydroxy alpha-carotene), a xanthophyll present in plant chloroplasts, increases the permeability of phospholipid vesicles to Ca2+, even though the pigment does not bind the metal ion. Energy-dependent uptake of Ca2+ by mitochondria is inhibited by lutein, which permits a rapid efflux of the ion from Ca2+-loaded mitochondria. These results are consistent with the view that the deleterious action of lutein on mitochondrial oxidative phosphorylation results from its destabilizing action on membrane structure.

Ionophore-mediated transmembrane movement of divalent cations in small unilamellar liposomes: An evaluation of the chlortetracycline fluorescence technique and correlations with black lipid membrane studies

Conceptual advances in the field of membrane transport have, in the main, utilized artificial membranes, both planar and vesicular. Systems of biological interest,viz., cells and organelles, resemble vesicles in size and geometry. Methods are, therefore, required to extend the results obtained with planar membranes to liposome systems. In this report we present an analysis of a fluorescence technique, using the divalent cation probe chlortetracycline, in small, unilamellar vesicles, for the study of divalent cation fluxes. An ion carrier (X537 A) and a pore former (alamethicin) have been studied. The rate of rise of fluorescence signal and the transmembrane ion gradient have been related to transmembrane current and potential, respectively. A second power dependence of ion conduction-including the electrically silent portion thereof — on X537 A concentration, has been observed. An exponential dependence of ldquocurrentrdquo on ldquotransmembrane potentialrdquo in the case of alamethicin is also confirmed. Possible errors in the technique are discussed.

Reaction product of lime and silica from rice husk ash

Rice husk ash (about 95% silica) with known physical and chemical characteristics has been reacted with lime and water. The setting process for a lime-excess and a lime-deficient mixture has been investigated. The product of the reaction has been shown to be a calcium silicate hydrate, C-S-H(I)+ by a combination of thermal analysis, XRD and electron microscopy. Formation of C-S-H(I) accounts for the strength of lime-rice husk ash cement.

Gel growth and dielectric studies of K. Rb and Cs perchlorates

Single crystals of K, Rb and Cs perchlorates have been grown by the counter diffusion of the respective ions and CIO4 through the gel medium. Studies on nucleation, growth kinetics, morphological aspects and purity are discussed in this paper. The dielectric constant, ~b, as well as loss measured along the longest axis, exhibits an anomaly at the transition temperature, Tt, in all the three crystals. It is found that the peak values of Tt are approximately 800, 100 and 53 in K, Rb and Cs perchlorates, respectively. The dielectric anomaly and the large value of c b in the cubic phase are discussed in terms of the degree of disorder of the CIO~ group and the possible contribution from defects.

A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide

Oxidation of NADH by rat erythrocyte plasma membrane was stimulated by about 50-fold on addition of decavanadate, but not other forms of vanadate like orthovanadate, metavanadate aad vanadyl sulphate. The vanadate-stimulated activity was observed only in phosphate buffer while other buffers like Tris, acetate, borate and Hepes were ineffective. Oxygen was consumed during the oxidation of NADH and the products were found to be NAD+ and hydrogen peroxide. The reaction had a stoichiometry of one mole of oxygen consumption and one mole of H2O2 production for every mole of NADH that was oxidized. Superoxide dismutase and manganous inhibited the activity indicating the involvement of superoxide anions. Electron spin resonance in the presence of a spin trap, 5, 5prime-dimethyl pyrroline N-oxide, indicated the presence of superoxide radicals. Electron spin resonance studies also showed the appearance of VIV species by reduction of VV of decavanadate indicating thereby participation of vanadate in the redox reaction. Under the conditions of the assay, vanadate did not stimulate lipid peroxidation in erythrocyte membranes. Extracts from lipid-free preparations of the erythrocyte membrane showed full activity. This ruled out the possibility of oxygen uptake through lipid peroxidation. The vanadate-stimulated NADH oxidation activity could be partially solubilized by treating erythrocyte membranes either with Triton X-100 or sodium cholate. Partially purified enzyme obtained by extraction with cholate and fractionation by ammonium sulphate and DEAE-Sephadex was found to be unstable.

Fluorescent alamethicin fragments A study of membrane activity and aqueous phase aggregation

The linear polypeptide antibiotic alamethicin is known to form channels in artificial lipid membranes. Synthetic 13- and 17-residue alamethicin fragments, labelled with a fluorescent dansyl group at the N-terminus, have been shown to translocate divalent cations across phospholipid membranes and to uncouple oxidative phosphorylation in rat liver mitochondria, in a manner analogous to the parent peptides. From studies of the aqueous phase aggregation behavior of the peptides, as well as their interaction with rat liver mitochondria, it is concluded that the interaction of the peptides with membranes is a complex process, probably involving both aqueous and membrane phase aggregation.

Silica from rice husk through thermal decomposition

The thermal decomposition characteristics of rice husk have been investigated by dynamic thermoanalytical techniques: DTA, TG, DTG and isothermal heating. The observed thermal behaviour is explained on the basis of a superposition of the decomposition of cellulose and lignin, which are the major organic constituents of rice husk. Morphological features of silica in husk as well as the ash are examined by scanning electron microscopy. Silica in the residual ash has been characterised by X-ray diffraction and infrared spectroscopy. Controlled thermal decomposition of rice husk has been shown to be a convenient method for the liberation of silica.

Forward osmosis as a pre-treatment for treating coal seam gas associated water: Flux and fouling behaviours

In this study, a bench scale forward osmosis (FO) process was operated using two commonly available FO membranes in different orientations in order to examine the removal of foulants in the coal seam gas (CSG) associated water, the water flux and fouling behaviours of the process were also investigated. After 48 h of fouling simulation experiment, the water flux declined by approximately 55 and 35% of its initial level in the TFC-PRO and CTA-PRO modes (support layer facing the feed), respectively, while the flux decline in the TFC-FO and CTA-FO modes (active layer facing the feed) was insignificant. The flux decline in PRO modes was caused by the compounding effects of internal concentration polarisation and membrane fouling. However, the declined flux was completely recovered to its initial level following the hydraulic cleaning using deionised water. Dissolved organic carbon (DOC), adenosine tri-phosphate (ATP) and major inorganic scalants (Ca, Mg and silica) in the CSG feed were effectively removed by using the FO process. The results of this study suggest that the FO process shows promising potential to be employed as an effective pre-treatment for membrane purification of CSG associated water.

Adsorption and release of biocides with mesoporous silica nanoparticles

In this proof-of-concept study, an agricultural biocide (imidacloprid) was effectively loaded into the mesoporous silica nanoparticles (MSNs) with different pore sizes, morphologies and mesoporous structures for termite control. This resulted in nanoparticles with a large surface area, tunable pore diameter and small particle size, which are ideal carriers for adsorption and controlled release of imidacloprid. The effect of pore size, surface area and mesoporous structure on uptake and release of imidacloprid was systematically studied. It was found that the adsorption amount and release profile of imidacloprid were dependent on the type of mesoporous structure and surface area of particles. Specifically, MCM-48 type mesoporous silica nanoparticles with a three dimensional (3D) open network structure and high surface area displayed the highest adsorption capacity compared to other types of silica nanoparticles. Release of imidacloprid from these nanoparticles was found to be controlled over 48 hours. Finally, in vivo laboratory testing on termite control proved the efficacy of these nanoparticles as delivery carriers for biopesticides. We believe that the present study will contribute to the design of more effective controlled and targeted delivery for other biomolecules.

Weakening of the Gram-negative bacterial outer membrane - A tool for increasing microbiological safety

Gram-negative bacteria are harmful in various surroundings. In the food industy their metabolites are potential cause of spoilage and this group also includes many severe or potential pathogens, such as Salmonella. Due to their ability to produce biofilms Gram-negative bacteria also cause problems in many industrial processes as well as in clinical surroundings. Control of Gram-negative bacteria is hampered by the outer membrane (OM) in the outermost layer of the cells. This layer is an intrinsic barrier for many hydrophobic agents and macromolecules. Permeabilizers are compounds that weaken OM and can thus increase the activity of antimicrobials by facililating entry of hydrophobic compounds and macromolecules into the cell where they can reach their target sites and inhibit or destroy cellular functions. The work described in this thesis shows that lactic acid acts as a permeabilizer and destabilizes the OM of Gram-negative bacteria. In addition, organic acids present in berriers, i.e. malic, sorbic and benzoic acid, were shown to weaken the OM of Gram-negative bacteria. Organic acids can poteniate the antimicrobial activity of other compounds. Microbial colonic degradation products of plant-derived phenolic compounds (3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3-phenylpropionic acid and 3-hydroxyphenylpropionic acid) efficiently destabilized OM of Salmonella. The studies increase our understanding of the mechanism of action of the classical chelator, ethylenediaminetetra-acetic acid (EDTA). In addition, the results indicate that the biocidic activity of benzalkonium chloride against Pseudomonas can be increased by combined use with polyethylenimine (PEI). In addition to PEI, several other potential permeabilizers, such as succimer, were shown to destabilize the OM of Gram-negative bacteria. Furthermore, combination of the results obtained from various permeability assays (e.g. uptake of a hydrophobic probe, sensitization to hydrophobic antibiotics and detergents, release of lipopolysaccharide (LPS) and LPS-specific fatty acids) with atomic force microscopy (AFM) image results increases our knowledge of the action of permeabilizers.

Immunologic studies on nucleic acids and their components. I. An analysis of the specificity of anti-deoxyadenylate antibodies by a membrane-binding technique

Anti-deoxyadenylate antibodies were produced in rabbits by injecting a conjugate of deoxyadenosine 5′-phosphate with bovine serum albumin. The antisera, as analyzed by double diffusion in agar and the quantitative precipitin reaction, showed hapten-specific antibodies. The specific interaction between [3H]deoxyadenylate and antiserum was studied by a sensitive nitrocellulose membrane-binding assay. The specificity of the antibodies was analyzed by measuring the effectiveness of other nucleotides or derivatives to inhibit the hapten-antibody binding. The requirements for recognition by the antibody sites were studied by using a series of naturally occurring nucleic acid components as well as some synthetic derivatives as inhibitors. The antibodies were found to show a high degree of specificity for the whole nucleotide, the base, sugar and phosphate playing almost equally important roles. There was cross reactivity with other mononucleotides, although of a low order. The antibodies were able to react with DNA and tRNA.

Paramagnetic probes in membrane biophysics

The use of paramagnetic probes in membrane research is reviewed. Electron paramagnetic resonance studies on model and biological membranes doped with covalent and non-covalent spin-labels have been discussed with special emphasis on the methodology and the type of information obtainable on several important phenomena like membrane fluidity, lipid flip-flop, lateral diffusion of lipids, lipid phase separation, lipid bilayer phase transitions, lipid-protein interactions and membrane permeability. Nuclear magnetic resonance spectroscopy has also been effectively used to study the conformations of cation mediators across membranes and to analyse in detail the transmembrane ionic motions at the mechanistic level.

Cu2+ inhibition of gel secretion in the xylem and its potential implications for water uptake of cut Acacia holosericea stems

Maintaining a high rate of water uptake is crucial for maximum longevity of cut stems. Physiological gel/tylosis formation decreases water transport efficiency in the xylem. The primary mechanism of action for post-harvest Cu2+ treatments in improving cut flower and foliage longevity has been elusive. The effect of Cu2+ on wound-induced xylem vessel occlusion was investigated for Acacia holosericea A. Cunn. ex G. Don. Experiments were conducted using a Cu2+ pulse (5 h, 2.2 mM) and a Cu2+ vase solution (0.5 mM) vs a deionized water (DIW) control. Development of xylem blockage in the stem-end region 10 mm proximal to the wounded stem surface was examined over 21 days by light and transmission electron microscopy. Xylem vessels of stems stood into DIW were occluded with gels secreted into vessel lumens via pits from surrounding axial parenchyma cells. Gel secretion was initiated within 1-2 days post-wounding and gels were detected in the xylem from day 3. In contrast, Cu2+ treatments disrupted the surrounding parenchyma cells, thereby inhibiting gel secretion and maintaining the vessel lumens devoid of occlusions. The Cu2+ treatments significantly improved water uptake by the cut stems as compared to the control. © 2013 Scandinavian Plant Physiology Society.

In vivo delivery of bovine viral diahorrea virus, E2 protein using hollow mesoporous silica nanoparticles

Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 [small mu ]g Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 [small mu ]g dose of E2 adsorbed to 250 [small mu ]g HMSA was compared to immunisation with Opti-E2 (50 [small mu ]g) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 [small mu ]g). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine.

Freeze-drying of ovalbumin loaded mesoporous silica nanoparticle vaccine formulation increases antigen stability under ambient conditions

Amino functionalised mesoporous silica nanoparticles (AM-41) have been identified as a promising vaccine delivery material. The capacity of AM-41 to stabilise vaccine components at ambient temperature (23–27 °C) was determined by adsorbing the model antigen ovalbumin (OVA) to AM-41 particles (OVA-41). The OVA-41 was successfully freeze-dried using the excipients 5% trehalose and 1% PEG8000. Both the immunological activity of OVA and the nanoparticle structure were maintained following two months storage at ambient temperature. The results of immunisation studies in mice with reconstituted OVA-41 demonstrated the induction of humoral and cell-meditated immune responses. The capacity of AM-41 particles to facilitate ambient storage of vaccine components without loss of immunological potency will underpin the further development of this promising vaccine delivery platform.